Mice expressing nonpolymerizable fibrinogen have reduced arterial and venous thrombosis with preserved hemostasis.

ABSTRACT: Elevated circulating fibrinogen levels correlate with increased risk for both cardiovascular and venous thromboembolic diseases. In vitro studies show that formation of a highly dense fibrin matrix is a major determinant of clot structure and stability. Here, we analyzed the impact of nonpolymerizable fibrinogen on arterial and venous thrombosis as well as hemostasis in vivo using FgaEK mice that express normal levels of a fibrinogen that cannot be cleaved by thrombin. In a model of carotid artery thrombosis, FgaWT/EK and FgaEK/EK mice were protected from occlusion with 4% ferric chloride (FeCl3) challenges compared with wild-type (FgaWT/WT) mice, but this protection was lost, with injuries driven by higher concentrations of FeCl3. In contrast, fibrinogen-deficient (Fga-/-) mice showed no evidence of occlusion, even with high-concentration FeCl3 challenge. Fibrinogen-dependent platelet aggregation and intraplatelet fibrinogen content were similar in FgaWT/WT, FgaWT/EK, and FgaEK/EK mice, consistent with preserved fibrinogen-platelet interactions that support arterial thrombosis with severe challenge. In an inferior vena cava stasis model of venous thrombosis, FgaEK/EK mice had near complete protection from thrombus formation. FgaWT/EK mice also displayed reduced thrombus incidence and a significant reduction in thrombus mass relative to FgaWT/WT mice after inferior vena cava stasis, suggesting that partial expression of nonpolymerizable fibrinogen was sufficient for conferring protection. Notably, FgaWT/EK and FgaEK/EK mice had preserved hemostasis in multiple models as well as normal wound healing times after skin incision, unlike Fga-/- mice that displayed significant bleeding and delayed healing. These findings indicate that a nonpolymerizable fibrinogen variant can significantly suppress occlusive thrombosis while preserving hemostatic potential in vivo.

Elimination of fibrin γ-chain cross-linking by FXIIIa increases pulmonary embolism arising from murine inferior vena cava thrombi.

The onset of venous thromboembolism, including pulmonary embolism, represents a significant health burden affecting more than 1 million people annually worldwide. Current treatment options are based on anticoagulation, which is suboptimal for preventing further embolic events. In order to develop better treatments for thromboembolism, we sought to understand the structural and mechanical properties of blood clots and how this influences embolism in vivo. We developed a murine model in which fibrin γ-chain cross-linking by activated Factor XIII is eliminated (FGG3X) and applied methods to study thromboembolism at whole-body and organ levels. We show that FGG3X mice have a normal phenotype, with overall coagulation parameters and platelet aggregation and function largely unaffected, except for total inhibition of fibrin γ-chain cross-linking. Elimination of fibrin γ-chain cross-linking resulted in thrombi with reduced strength that were prone to fragmentation. Analysis of embolism in vivo using Xtreme optical imaging and light sheet microscopy demonstrated that the elimination of fibrin γ-chain cross-linking resulted in increased embolization without affecting clot size or lysis. Our findings point to a central previously unrecognized role for fibrin γ-chain cross-linking in clot stability. They also indirectly indicate mechanistic targets for the prevention of thrombosis through selective modulation of fibrin α-chain but not γ-chain cross-linking by activated Factor XIII to reduce thrombus size and burden, while maintaining clot stability and preventing embolism.

GPVI (Glycoprotein VI) Interaction With Fibrinogen Is Mediated by Avidity and the Fibrinogen αC-Region.

OBJECTIVE: GPVI (glycoprotein VI) is a key molecular player in collagen-induced platelet signaling and aggregation. Recent evidence indicates that it also plays important role in platelet aggregation and thrombus growth through interaction with fibrin(ogen). However, there are discrepancies in the literature regarding whether the monomeric or dimeric form of GPVI binds to fibrinogen at high affinity. The mechanisms of interaction are also not clear, including which region of fibrinogen is responsible for GPVI binding. We aimed to gain further understanding of the mechanisms of interaction at molecular level and to identify the regions on fibrinogen important for GPVI binding. Approach and Results: Using multiple surface- and solution-based protein-protein interaction methods, we observe that dimeric GPVI binds to fibrinogen with much higher affinity and has a slower dissociation rate constant than the monomer due to avidity effects. Moreover, our data show that the highest affinity interaction of GPVI is with the αC-region of fibrinogen. We further show that GPVI interacts with immobilized fibrinogen and fibrin variants at a similar level, including a nonpolymerizing fibrin variant, suggesting that GPVI binding is independent of fibrin polymerization. CONCLUSIONS: Based on the above findings, we conclude that the higher affinity of dimeric GPVI over the monomer for fibrinogen interaction is achieved by avidity. The αC-region of fibrinogen appears essential for GPVI binding. We propose that fibrin polymerization into fibers during coagulation will cluster GPVI through its αC-region, leading to downstream signaling, further activation of platelets, and potentially stimulating clot growth. Graphic Abstract: A graphic abstract is available for this article.

Patients with paroxysmal nocturnal hemoglobinuria demonstrate a prothrombotic clotting phenotype which is improved by complement inhibition with eculizumab.

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hematological disorder, characterized by complement-mediated intravascular hemolysis and thrombosis. The increased incidence of PNH-driven thrombosis is still poorly understood, but unlike other thrombotic disorders, is thought to largely occur through complement-mediated mechanisms. Treatment with a C5 inhibitor, eculizumab, has been shown to significantly reduce the number of thromboembolic events in these patients. Based on previously described links between changes in fibrin clot structure and thrombosis in other disorders, our aim was to investigate clot structure as a possible mechanism of thrombosis in patients with PNH and the anti-thrombotic effects of eculizumab treatment on clot structure. Clot structure, fibrinogen levels and thrombin generation were examined in plasma samples from 82 patients from the National PNH Service in Leeds, UK. Untreated PNH patients were found to have increased levels of fibrinogen and thrombin generation, with subsequent prothrombotic changes in clot structure. No link was found between increasing disease severity and fibrinogen levels, thrombin generation, clot formation or structure. However, eculizumab treated patients showed decreased fibrinogen levels, thrombin generation and clot density, with increasing time spent on treatment augmenting these antithrombotic effects. These data suggest that PNH patients have a prothrombotic clot phenotype due to increased fibrinogen levels and thrombin generation, and that the antithrombotic effects of eculizumab are, in-part, due to reductions in fibrinogen and thrombin generation with downstream effects on clot structure.

Breast radiology malpractice suits: Characteristics of frequency and outcomes- national and statewide distinctions.

The purpose of this study was to determine the frequency and outcomes of breast malpractice suits among all enrollees in One Call Medical Inc.'s panel of interpreting radiologists nationally and to evaluate state-by-state variations. The 8401 radiologists enrolled had 4764 suits, of which 826 were related to breast disease. In New York and New Jersey, the ratio of breast suits to One Call Radiologists was 0.28 and 0.27, twice as much as in any other state. Breast suits in radiology have a wide variation in frequency across the country with New York and New Jersey far exceeding all others in relative frequency and number of radiologists with multiple breast suits.

Health literacy and online educational resources: an opportunity to educate patients.

OBJECTIVE: Given the increasing accessibility of material on the Internet and the use of these materials by patients as a source of health care information, the purpose of this study was to quantitatively evaluate the level of readability of resources made available on the European Society of Radiology website to determine whether these materials meet the health literacy needs of the general public as set forth by guidelines of the U.S. National Institutes of Health (NIH) and the American Medical Association (AMA). MATERIALS AND METHODS: All 41 patient education articles created by the European Society of Radiology (ESR) were downloaded and analyzed with the following 10 quantitative readability scales: the Coleman-Liau Index, Flesch-Kincaid Grade Level, Flesch Reading Ease, FORCAST Formula, Fry Graph, Gunning Fog Index, New Dale-Chall, New Fog Count, Raygor Reading Estimate, and the Simple Measure of Gobbledygook. RESULTS: The 41 articles were written collectively at a mean grade level of 13.0 ± 1.6 with a range from 10.8 to 17.2. For full understanding of the material, 73.2% of the articles required the reading comprehension level of, at minimum, a high school graduate (12th grade). CONCLUSION: The patient education resources on the ESR website are written at a comprehension level well above that of the average Internet viewer. The resources fail to meet the NIH and AMA guidelines that patient education material be written between the third and seventh grade levels. Recasting these resources in a simpler format would probably lead to greater comprehension by ESR website viewers.

Radiology medical malpractice suits in gastrointestinal radiology: prevalence, causes, and outcomes.

The purpose of this study is to determine the prevalence, causes, and outcomes of GI malpractice suits in a survey of 8,401 radiologists. The malpractice histories of 8,401 radiologists from 47 states were evaluated from credentialing data of all radiologists participating in the network of One Call Medical Inc. Thirty-two percent of radiologists were defendants in at least one malpractice suit. Of the 4,073 total claims, 346 (8.49 %) were related to the gastrointestinal system. The most frequent primary allegations were failure to diagnose, 65.9 %, and procedural complications, 17.1 %. The commonest missed diagnoses were malignancy, 31.6 %; pneumoperitoneum, 19.3 %; and appendicitis, 14.5 %. Payment to the plaintiff occurred in 75.8 % of claims pertinent to cancer, 73.2 % for missed pneumoperitoneum, and 62.5 % related to appendicitis. Of cases in which a ruling was made in favor of the plaintiff, median payments for pneumoperitoneum was $215,000, for primary cancer $200,000, and for appendicitis $60,000. Among procedurally related errors resulting in judgment against a defending radiologist, 78.6 % of claims regarding retained foreign body, 75 % of barium enema cases, and 62.5 % of liver biopsy resulted in a payment to the plaintiff. Among all resolved cases, the median award was $30,000 for unrecognized foreign body retention, $100,000 for barium enema complications, and $400,000 for liver biopsy complication. Of all GI malpractice claims, failure to diagnose was the most prevalent. Among them, approximately three fourths of claims related to either the diagnosis of primary cancer or for detection of a pneumoperitoneum.

A critical review of the readability of online patient education resources from RadiologyInfo.Org.

OBJECTIVE: Health consumers and their families rely on the Internet as a source of authoritative information regarding the procedures used to reach a diagnosis, effect treatment, and influence prognosis. In radiology, online materials can be a means by which to offer patients comprehensible explanations of the capabilities, the risks and rewards, and the techniques under our purview. Consequently, estimations of health literacy should take into account the reading level of the average American when composing and transmitting such information to the lay public without the mediation of a referring physician. MATERIALS AND METHODS: In December 2012, patient education reports from the files of RadiologyInfo.org, a jointly sponsored website of the American College of Radiology and the Radiological Society of North America, were downloaded to assess their textual sophistication. All 138 patient education articles including the glossary were analyzed for their respective level of "readability" using the following 10 evaluative scales: Flesch Reading Ease, Flesch-Kincaid Grade Level, Simple Measure of Gobbledygook Grading, Coleman-Liau Index, Gunning Fog Index, New Dale-Chall scale, FORCAST, Fry graph, Raygor Readability Estimate, and New Fog Count. RESULTS: The 138 online patient education articles were written, on average, between the 10th and 14th grade levels, which exceeds both the American Medical Association and the National Institutes of Health recommendations that patient education resources be comprehensible to those who read no higher than the seventh grade level. CONCLUSION: Patients may accrue a greater benefit from written articles available on RadiologyInfo.org if the texts were revised to be in compliance with the National Institutes of Health and American Medical Association grade level recommendations. This could lead to a broadened appreciation of the capabilities of radiology's role in general and enhanced understanding of imaging techniques and their application to clinical practice.

Non-spinal musculoskeletal malpractice suits against radiologists in the USA--rates, anatomic locations, and payments in a survey of 8,265 radiologists.

The objective of this study was to present the characteristics of non-spinal musculoskeletal malpractice suits with attention to rates, anatomic location, and payments in a survey of 8,265 radiologists. The malpractice histories of 8,265 radiologists from 36 states were evaluated from credentialing data required of all radiologists participating in the network of One Call Medical, Inc., a broker for imaging tests in workmen's compensation cases. Twenty six hundred of the 8,265 radiologists (31.5 %) had at least one suit. Of the 4,741 total claims, 627 (13.2 %) were related to the bones and soft tissues. Four hundred seventeen (66.1 %) of them involved the musculoskeletal system other than the spine. A cause was known for 400. Of these, 91.8 % (367/400) resulted from an alleged failure to diagnose. The foot was the most common site with a rate 6.00 cases/1,000 radiologist's person years (95 % confidence interval (CI), 4.68-7.68), and the hip was second with a rate of 5.30 cases/1,000 person years (95 % CI, 4.15-6.76). The highest median payment related to ankle injuries with a median settlement of $72,500 (interquartile range (IQR), $40,000-$161,250). The state in which the highest median settlement occurred was Maryland ($125,000; IQR, $95,000-$230,000)) whereas Utah had the highest rate of suits (5.24 cases per 1,000 person years; CI, 3.03-9.04). Claims regarding foot and hip injury were the most common, but ankle settlements incurred the highest awards.

Nitrosyl Hemoglobin Formation from Nitrite in Normal and Sickle Blood.

Sickle cell anemia is caused by a single mutation in the gene encoding the beta subunit of hemoglobin. Due to this mutation, sickle cell hemoglobin (HbS) polymerizes under hypoxic conditions, decreasing red blood cell deformability and leading to multiple pathological effects that cause substantial morbidity and mortality. Several pre-clinical and human studies have demonstrated that the anion nitrite has potential therapeutic benefits for patients with sickle cell disease. Nitrite is reduced to nitric oxide (NO) by deoxygenated hemoglobin contributing to vasodilation, decreasing platelet activation, decreasing cellular adhesion to activated endothelium, and decreasing red cell hemolysis; all of which could ameliorate patient morbidities. Previous work on extracellular hemoglobin has shown that solution phase HbS reduces nitrite to NO faster than normal adult hemoglobin (HbA), while polymerized HbS reduces nitrite slower than HbA. In this work, we compared the rate of nitrite reduction to NO measured by the formation of nitrosyl hemoglobin in sickle and normal red blood cells at varying hemoglobin oxygen saturations. We found the overall rate of nitrite reduction between normal and sickle red blood cells was similar and confirmed this result under partially oxygenated conditions, but normal red blood cells reduced nitrite faster than sickle red blood cells under anoxia where HbS polymerization is maximal. These results are consistent with previous work using extracellular hemoglobin where the rate of reduction by solution phase HbS makes up for the slower reduction by polymer phase HbS under partially oxygenated conditions, but the polymer phase kinetics dominates in the complete absence of oxygen.

The causes of medical malpractice suits against radiologists in the United States.

PURPOSE: To determine the most frequent causes of malpractice suits as derived from credentialing data of 8401 radiologists. MATERIALS AND METHODS: This study was approved by the Institutional Review Board of New Jersey Medical School. A total of 8401 radiologists in 47 states participating in the network of One-Call Medical, a broker for computed tomographic/magnetic resonance studies in workers' compensation cases, were required to provide their malpractice history as part of their credentialing application. Of these, 2624 (31%) radiologists had at least one claim in their career. In each enrollee's credentialing file, if there was a claim against the enrollee there was a narrative regarding each malpractice case from which, in most instances, a primary allegation could be discerned. Among the 4793 cases, an alleged cause could be derived from the narrative in 4043 (84%). Statistical analysis was performed with Stata 12 (2011; Stata, College Station, Tex) software. RESULTS: The most common general cause was error in diagnosis (14.83 claims per 1000 person-years [95% confidence interval {CI}: 14.19, 15.51]). In this category, breast cancer was the most frequently missed diagnosis (3.57 claims per 1000 person-years [95% CI: 3.26, 3.91]), followed by nonspinal fractures (2.49 claims per 1000 person-years [95% CI: 2.28, 2.72]), spinal fractures (1.32 claims per 1000 person-years [95% CI: 1.16, 1.49]), lung cancer (1.26 claims per 1000 person-years [95% CI: 1.11, 1.42]), and vascular disease (1.08 claims per 1000 person-years [95% CI: 0.93, 1.24]). The category next in frequency was procedural complications (1.76 claims per 1000 person-years [95% CI: 1.58, 1.96]), followed by inadequate communication with either patient (0.40 claim per 1000 person-years [95% CI: 0.32, 0.50]) or referrer (0.71 claim per 1000 person-years [95% CI: 0.60, 0.84]). Radiologists had only a peripheral role in 0.92 claim per 1000 person-years (95% CI: 0.77, 1.10). Failure to recommend additional testing was a rare cause (0.41 claim per 1000 person-years [95% CI: 0.34, 0.50]). CONCLUSION: Errors in diagnosis are, by far, the most common generic cause of malpractice suits against radiologists. In this category, breast cancer was the most frequently missed diagnosis, followed by nonvertebral fractures and spinal fractures. Failure to communicate and failure to recommend additional testing are both uncommon reasons for initiating a suit.

The demography of medical malpractice suits against radiologists.

PURPOSE: To delimit demographic characteristics of malpractice claims against radiologists in the United States by sex and location and to note the varying percentages of favorable outcomes and award amounts to plaintiffs by state. MATERIALS AND METHODS: This HIPAA-compliant study was institutional review board-approved. All radiologists enrolled in One-Call Medical, a specialized preferred provider organization, go through an initial and recurrent credentialing process, which records state of residence, age, sex, and malpractice history. For each radiologist, a record of unfavorable outcomes and payment awards is derived from narratives provided by the National Practitioner's Data Bank. All other suits are self-reported. Rates of malpractice claims per state were calculated with a zero-inflated negative binomial regression model allowing for differences in years at risk. Poisson regression was used to calculate the incidence rate ratio (IRR) for any payment as a result of a malpractice claim compared with the average of all 36 states, adjusted for sex. To determine the association of age, sex, and state with amount of payment, we used a general linear model assuming a gamma family distribution. RESULTS: In August 2010, 8401 radiologists from 47 states composed One-Call Medical's panel. During their careers, 30.9% (2600 of 8401) had been the subject of at least one malpractice claim. Median payment awards varied by 14-fold from Maine ($350 000) to Colorado ($24 105), while mean payments varied ninefold from Oregon ($715 707) to Nebraska ($74 373). Adjusted for age and state, radiologists in Alabama had the lowest rate of malpractice suits per 100 practice-years for men (0.95; 95% confidence interval [CI]; 0.73, 1.28) and women (0.70; 95% CI: 0.52, 0.96) compared with those in New York, who had the highest rate of suits for men (5.65; 95% CI: 5.09, 6.26) and women (4.13; 95% CI: 3.54, 4.80). Overall, male radiologists had a higher rate of being sued than did their female counterparts (IRR = 1.37; 95% CI: 1.20, 1.56). CONCLUSION: The likelihood of a radiologist being the defendant in at least one suit is 50% by age 60, yet the difference in frequency and average number of suits accrued varies widely by state of residence and sex. Among resolved suits, the percentage of cases in which payment was made to the plaintiff differs markedly by state, as do median and mean award amount. Men are more likely than women to be sued during the course of their careers.

Spinal-related malpractice suits against radiologists in the USA-rates, anatomic location, percent of adverse judgments, and average payments.

To present overall rates, anatomic location, percent of adverse settlements to the radiologists, and average payments to the plaintiff in spinal-related malpractice suits in a survey of 8,265 radiologists. The malpractice histories of 8,265 radiologists from 36 states were evaluated from credentialing data required of all radiologists participating in the network of One Call Medical Incorporated, a broker for CT/MR in workmen's compensation cases. Two hundred twenty-six of the 8,265 radiologists (31.5 %) had at least one suit. Of the 4,741 total claims, 627 (13.2 %) were related to the bones and adjacent soft tissue. Two hundred and ten (32.9 %) involved the spine. Of these, 70.2 % (134/191) were settled in favor of the plaintiff. One hundred and sixteen (68.2 %) involved the cervical spine with an average settlement of $483,156. Lumbar cases accounted for 28 (16.5 %) of spinal suits, with an average settlement of $119,272. Thoracic cases (26) accounted for only 15.3 % of spinal cases and had an average settlement of $481,608. An allegation of spinal malpractice resulting in a settlement or judgment against the radiologist occurred at a rate of 29.5 cases per 1,000 radiologists' person years. Of the three spinal regions, the cervical spine was the most frequent anatomic site of a malpractice suit and among all those cases settled incurred the highest payment in judgment to the plaintiff.

Plasma from patients with pulmonary embolism show aggregates that reduce after anticoagulation.

BACKGROUND: Microclots, a term also used for amyloid fibrin(ogen) particles and henceforth named aggregates, have recently been reported in the plasma of patients with COVID-19 and long COVID. These aggregates have been implicated in the thrombotic complications of these diseases. METHODS: Plasma samples from 35 patients with acute pulmonary embolism were collected and analysed by laser scanning confocal microscopy and scanning electron microscopy before and after clotting. RESULTS: Here we confirm the presence of aggregates and show that they also occur in the plasma of patients with pulmonary embolism, both before and after clotting. Aggregates vary in size and consist of fibrin and platelets. We show that treatment with low-molecular weight heparin reduces aggregates in the samples of patients with pulmonary embolism. Double centrifugation of plasma does not eliminate the aggregates. CONCLUSIONS: These data corroborate the existence of microclots or aggregates in diseases associated with venous thromboembolism. Important questions are raised regarding their pathophysiological relevance and further studies are warranted to investigate whether they represent cause or consequence of clinical thrombosis.

When blood turns from liquid to solid, a protein called fibrin and cells called platelets aggregate to form a blood clot. Small aggregates have been found in the blood of people with COVID-19 and long COVID. Here, we show that small aggregates also occur in the blood of patients with pulmonary embolism, a disorder in which blood clots are trapped in an artery in the lung, preventing blood flow. We confirm that aggregates consist of fibrin and platelets, and show that the number of aggregates is lower when patients are treated with blood thinning drugs. These results suggest other disorders of the blood should also be investigated to see whether aggregates are present and whether they have an impact on the outcome for the patient. This could help us understand the cause of diseases associated with blood clotting, which might offer new approaches for diagnosis and treatment.

Relative importance of metaphor in radiology versus other medical specialties.

The acquisition of competence in radiology often entails referring to other realms of knowledge, by which insights are acquired through the use of metaphor. One way in which compelling associations are made and retained is by linking anatomic structures and pathologic conditions with objects, places, and concepts, and codifying these relationships as metaphoric signs. An aggregate of specialty-specific signs were obtained from two general medical dictionaries and from encyclopedic texts in radiology and six other specialties: internal medicine, dermatology, pathology, general surgery, orthopedics, and pediatrics. The signs were then separated into two categories: eponymous (bearing the name of an individual or place) and metaphoric (extending meaning from one context to another). A total of 375 metaphoric signs were collected from citations in the researched dictionaries and texts, the overwhelming majority (66%) of which were radiologic in reference. In every other specialty, eponymous signs outnumbered metaphoric signs. In contrast, eponymous signs were comparatively infrequent in radiology. The striking difference observed in the data highlights the importance of metaphors for discourse and instruction in radiology. In image interpretation, the meaning of perceptual input is often discerned through associations with pictures previously encountered and understood both concretely and metaphorically. The inherent nature of radiologic images as simulacra of both normal anatomy and disease entities makes imaging findings well suited to explanation by means of named patterns borrowed from other realms of knowledge.

Three-dimensional tracking using a single-spot rotating point spread function created by a multiring spiral phase plate.

Significance: Three-dimensional (3D) imaging and object tracking is critical for medical and biological research and can be achieved by multifocal imaging with diffractive optical elements (DOEs) converting depth ( z ) information into a modification of the two-dimensional image. Physical insight into DOE designs will spur this expanding field. Aim: To precisely track microscopic fluorescent objects in biological systems in 3D with a simple low-cost DOE system. Approach: We designed a multiring spiral phase plate (SPP) generating a single-spot rotating point spread function (SS-RPSF) in a microscope. Our simple, analytically transparent design process uses Bessel beams to avoid rotational ambiguities and achieve a significant depth range. The SPP was inserted into the Nomarski prism slider of a standard microscope. Performance was evaluated using fluorescent beads and in live cells expressing a fluorescent chromatin marker. Results: Bead localization precision was < 25 nm in the transverse dimensions and ≤ 70 nm along the axial dimension over an axial range of 6 µ m . Higher axial precision ( ≤ 50 nm ) was achieved over a shallower focal depth of 2.9 µ m . 3D diffusion constants of chromatin matched expected values. Conclusions: Precise 3D localization and tracking can be achieved with a SS-RPSF SPP in a standard microscope with minor modifications.

Fibrin protofibril packing and clot stability are enhanced by extended knob-hole interactions and catch-slip bonds.

Fibrin polymerization involves thrombin-mediated exposure of knobs on one monomer that bind to holes available on another, leading to the formation of fibers. In silico evidence has suggested that the classical A:a knob-hole interaction is enhanced by surrounding residues not directly involved in the binding pocket of hole a, via noncovalent interactions with knob A. We assessed the importance of extended knob-hole interactions by performing biochemical, biophysical, and in silico modeling studies on recombinant human fibrinogen variants with mutations at residues responsible for the extended interactions. Three single fibrinogen variants, γD297N, γE323Q, and γK356Q, and a triple variant γDEK (γD297N/γE323Q/γK356Q) were produced in a CHO (Chinese Hamster Ovary) cell expression system. Longitudinal protofibril growth probed by atomic force microscopy was disrupted for γD297N and enhanced for the γK356Q mutation. Initial polymerization rates were reduced for all variants in turbidimetric studies. Laser scanning confocal microscopy showed that γDEK and γE323Q produced denser clots, whereas γD297N and γK356Q were similar to wild type. Scanning electron microscopy and light scattering studies showed that fiber thickness and protofibril packing of the fibers were reduced for all variants. Clot viscoelastic analysis showed that only γDEK was more readily deformable. In silico modeling suggested that most variants displayed only slip-bond dissociation kinetics compared with biphasic catch-slip kinetics characteristics of wild type. These data provide new evidence for the role of extended interactions in supporting the classical knob-hole bonds involving catch-slip behavior in fibrin formation, clot structure, and clot mechanics.

Quantitative analysis of clot density, fibrin fiber radius, and protofibril packing in acute phase myocardial infarction.

INTRODUCTION: Coronary artery disease is associated with impaired clot structure. The aim of this study was to investigate acute phase myocardial infarction (AMI) and provide detailed quantitative analysis of clot ultrastructure. MATERIALS AND METHODS: Clot formation and breakdown, pore size, fiber density, fiber radius and protofibril packing were investigated in plasma clots from AMI patients. These data were compared to those from healthy controls. RESULTS: Analysis on clot formation using turbidity showed increased lag time, suggesting changes in protofibril packing and increased fiber size for AMI patients compared to healthy controls. Additionally, increased average rate of clotting and decreased time to maximum absorbance in AMI patients suggest that clots formed more quickly. Moreover, we observed increased time from max OD to max rate of lysis. Increased fibrinogen and decreased plasminogen in AMI patients were accounted for in represented significant differences. AMI samples showed increased time to 25% and 50% lysis, but no change in 75% lysis, representative of delayed lysis onset, but expediated lysis once initiated. These data suggest that AMI patients formed less porous clots made from more densely packed fibers with decreased numbers of protofibrils, which was confirmed using decreased permeation and increased fiber density, and decreased turbidimetry. CONCLUSIONS: AMI plasma formed clots that were denser, less permeable, and lysed more slowly than healthy controls. These findings were confirmed by detailed analysis of clot ultrastructure, fiber size, and protofibril packing. Dense clot structures that are resistant to lysis may contribute to a prothrombotic milieu in AMI.

Automated Fiber Diameter and Porosity Measurements of Plasma Clots in Scanning Electron Microscopy Images.

Scanning Electron Microscopy (SEM) is a powerful, high-resolution imaging technique widely used to analyze the structure of fibrin networks. Currently, structural features, such as fiber diameter, length, density, and porosity, are mostly analyzed manually, which is tedious and may introduce user bias. A reliable, automated structural image analysis method would mitigate these drawbacks. We evaluated the performance of DiameterJ (an ImageJ plug-in) for analyzing fibrin fiber diameter by comparing automated DiameterJ outputs with manual diameter measurements in four SEM data sets with different imaging parameters. We also investigated correlations between biophysical fibrin clot properties and diameter, and between clot permeability and DiameterJ-determined clot porosity. Several of the 24 DiameterJ algorithms returned diameter values that highly correlated with and closely matched the values of the manual measurements. However, optimal performance was dependent on the pixel size of the images-best results were obtained for images with a pixel size of 8-10 nm (13-16 pixels/fiber). Larger or smaller pixels resulted in an over- or underestimation of diameter values, respectively. The correlation between clot permeability and DiameterJ-determined clot porosity was modest, likely because it is difficult to establish the correct image depth of field in this analysis. In conclusion, several DiameterJ algorithms (M6, M5, T3) perform well for diameter determination from SEM images, given the appropriate imaging conditions (13-16 pixels/fiber). Determining fibrin clot porosity via DiameterJ is challenging.

Fibrinogen αC-subregions critically contribute blood clot fibre growth, mechanical stability, and resistance to fibrinolysis.

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.