Cryoneurolysis of the Femoral Nerve for Focal Spasticity in an Ambulatory Patient.

Introduction: Spasticity of the knee extensors is a common presentation among patients with multiple sclerosis. The resulting stiff leg gait can result in increased risk of falls, heightened energy expenditure during gait, lowered gait speed, and compensatory gait mechanisms that increase wear on the hips. Cryoneurolysis is a novel percutaneous, minimally invasive treatment for focal spasticity. Methods: A single patient with multiple sclerosis was treated with cryoneurolysis of the femoral nerve branch to rectus femoris. The patient was followed for 15 months. Spasticity severity, gait speed, and patient reported outcomes were collected at each follow-up. Results: Spasticity severity as per the Modified Ashworth Scale was reduced at 1 month, with change persisting up to 15 months post-procedure. Range of motion as per the Modified Tardieu Scale showed gradual improvement over the 15-month period. Gait speed increased after the procedure from 21.15 seconds to 12.49 seconds for the 10 m walk test 1 month post-procedure, then slowed to baseline after 15 months. The patient's confidence in their gait improved and their independence was maintained throughout the follow-up period. Because of the regression in the 10 m walk test, the patient elected to have the procedure repeated after 15 months. Immediately after the procedure, the 10 m test time improved to 16.20 seconds. Conclusion: Cryoneurolysis of the femoral nerve may be an effective, long-lasting treatment for spasticity causing stiff knee gait in patients with multiple sclerosis.

A Case Report of Cryoneurolysis for Dorsal Foot Pain and Toe Clawing in a Patient With Multiple Sclerosis.

Toe clawing in patients with upper motor neuron disorders is often attributed to the flexor digitorum longus (FDL) and is a common presentation among patients with multiple sclerosis (MS). This movement may be painful because of the altered pressure distribution and may increase the risk of falls, heighten energy expenditure during gait, and lower gait speed. Cryoneurolysis is a minimally invasive treatment that may be beneficial for pain and focal muscle hypertonicity. An ambulatory patient with MS was treated bilaterally with cryoneurolysis to the superficial fibular nerves for pain on the dorsum of the foot, and to the intramuscular tibial nerve motor branch to FDL for toe clawing. The patient felt that toe clawing was immediately reduced during gait and noted the ability to voluntarily spread their toes. The patient stated that the neuropathic pain on the dorsum of the foot was fully eliminated immediately post procedure. The patient reported improved confidence in their gait, maintained independence, and reduced toe clawing during a structured interview 12 weeks after treatment. The effects lasted for 5.5 months before symptoms returned. Retreatment at 6 months reproduced the benefits. The patient reported a positive experience with cryoneurolysis for toe clawing and dorsal foot pain.

Thrombin cleavage of the hepatitis E virus polyprotein at multiple conserved locations is required for genome replication.

The genomes of positive-sense RNA viruses encode polyproteins that are essential for mediating viral replication. These viral polyproteins must undergo proteolysis (also termed polyprotein processing) to generate functional protein units. This proteolysis can be performed by virally-encoded proteases as well as host cellular proteases, and is generally believed to be a key step in regulating viral replication. Hepatitis E virus (HEV) is a leading cause of acute viral hepatitis. The positive-sense RNA genome is translated to generate a polyprotein, termed pORF1, which is necessary and sufficient for viral genome replication. However, the mechanism of polyprotein processing in HEV remains to be determined. In this study, we aimed to understand processing of this polyprotein and its role in viral replication using a combination of in vitro translation experiments and HEV sub-genomic replicons. Our data suggest no evidence for a virally-encoded protease or auto-proteolytic activity, as in vitro translation predominantly generates unprocessed viral polyprotein precursors. However, seven cleavage sites within the polyprotein (suggested by bioinformatic analysis) are susceptible to the host cellular protease, thrombin. Using two sub-genomic replicon systems, we demonstrate that mutagenesis of these sites prevents replication, as does pharmacological inhibition of serine proteases including thrombin. Overall, our data supports a model where HEV uses host proteases to support replication and could have evolved to be independent of a virally-encoded protease for polyprotein processing.

Analysis of Adverse Effects of Cryoneurolysis for the Treatment of Spasticity.

OBJECTIVE: The aim of the study is to report adverse effects from clinical studies on ultrasound-guided percutaneous cryoneurolysis for spasticity. DESIGN: Patients were prospectively enrolled in three studies at a single institution. Cryoneurolysis was performed to primarily motor nerve branches (medial and lateral pectoral, musculocutaneous, radial, median, ulnar, tibial, obturator) and mixed motor sensory nerve trunks (median, ulnar, suprascapular, radial, and tibial). RESULTS: Cryoneurolysis was performed for 277 nerves (99 mixed motor sensory), on 113 patients (59 F, 54 M, average age 54.4 yrs). One patient had a local skin infection and two patients had bruising or swelling; all resolved within 1 mo. Nine reported nerve pain or dysesthesia (two motor, seven mixed motor sensory nerves). Four received no treatment, four oral or topical medications, two perineural injections, one botulinum toxin. Three patients' symptoms remained until 3 mos and one had numbness at six. One patient had botulinum toxin injections for cramping. All had at minimum 3-mo follow-up; seven withdrew (x̄ = 5.4 mos), four passed away. None of these 11 reported adverse effects. CONCLUSIONS: A total of 96.75% of nerve treatments had no pain or dysesthesias beyond treatment. Few had pain or numbness beyond 3 mos. Cryoneurolysis has potential to be a safe spasticity treatment with manageable adverse effects.

Bilateral Suprascapular Nerve Cryoneurolysis for Pain Associated With Glenohumeral Osteoarthritis: A Case Report.

Osteoarthritis is a leading cause of disability, typically treated with exercise, analgesics, injections, or surgeries. Cryoneurolysis is an established technique for the treatment of pain, including osteoarthritis that may provide an alternative for patients in whom surgery is not appropriate and conservative measures have failed. We present our experience with a 78-year-old man with severe pain from bilateral glenohumeral osteoarthritis. Their condition is complicated by several concurrent diagnoses, leaving them ineligible for surgical intervention, despite pharmacologic treatments proving insufficient to manage their pain. As an alternative, bilateral cryoneurolysis of the suprascapular nerve was performed at the suprascapular notch. Pain and disability scores both lessened on the Brief Pain Inventory Score, Disabilities of the Arm Shoulder and Hand (change of 9 points after 170 days) as well as the Shoulder Pain and Disability Index (change of 19 points after 170 days). The patient had improved active and passive range of motion for flexion, abduction, and external rotation of the shoulder. Improvements endured to follow-up at 170 days. There were no negative side effects as a result of the procedure.

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.

Fibrin-glycoprotein VI interaction increases platelet procoagulant activity and impacts clot structure.

BACKGROUND: The glycoprotein VI (GPVI) signaling pathway was previously reported to direct procoagulant platelet activity through collagen binding. However, the impact of GPVI-fibrin interaction on procoagulant platelet development and how it modulates the clot structure are unknown. OBJECTIVES: To determine the effect of GPVI-fibrin interaction on the platelet phenotype and its impact on the clot structure. METHODS: Procoagulant platelets in platelet-rich plasma clots were determined by scanning electron microscopy (wild-type and GPVI-deficient murine samples) and confocal microscopy. Procoagulant platelet number, clot density, clot porosity, and clot retraction were determined in platelet-rich plasma or whole blood clots of healthy volunteers in the presence of tyrosine kinase inhibitors (PRT-060318, ibrutinib, and dasatinib) and eptifibatide. RESULTS: GPVI-deficient clots showed a higher nonprocoagulant vs procoagulant platelet ratio than wild-type clots. The fiber density and the procoagulant platelet number decreased in the presence of Affimer proteins, inhibiting GPVI-fibrin(ogen) interaction and the tyrosine kinase inhibitors. The effect of GPVI signaling inhibitors on the procoagulant platelet number was exacerbated by eptifibatide. The tyrosine kinase inhibitors led to an increase in clot porosity; however, no differences were observed in the final clot weight, following clot retraction with the tyrosine kinase inhibitors, except for ibrutinib. In the presence of eptifibatide, clot retraction was impaired. CONCLUSION: Our findings showed that GPVI-fibrin interaction significantly contributes to the development of procoagulant platelets and that inhibition of GPVI signaling increases clot porosity. Clot contractibility was impaired by the integrin αIIbß3 and Btk pathway inhibition. Thus, inhibition of GPVI-fibrin interactions can alleviate structural characteristics that contribute to a prothrombotic clot phenotype, having potential important implications for novel antithrombotic interventions.

A retrospective review of the management and outcomes of patients diagnosed with complex regional pain syndrome type II using electrodiagnostic findings.

Objectives: The objective of this study was to assess the outcomes of the use of electrodiagnosis in the diagnosis and management of discrete nerve injuries in patients with complex regional pain syndrome (CRPS). Design: This study is a secondary retrospective cohort analysis of patients diagnosed with CRPS from a single outpatient physical medicine and rehabilitation clinic and included all patients who had abnormal electrodiagnostic findings, in addition to CRPS. Results: Sixty patients of 248 diagnosed with CRPS underwent electrodiagnosis, 41 of whom had abnormal electrodiagnostic findings indicating a discrete nerve injury. Only 51% of the 41 referrals had indicated the suspicion of a nerve injury. Nearly all patients had undergone physiotherapy. Forty-one percent responded to treatment with oral prednisone alone, 54% had a functional improvement after a combination of treatments including corticosteroids, and 5% improved with treatments that did not involve corticosteroids. Surgical interventions for nerve injuries were required for 34% of patients in the cohort. All surgeries involved the median or ulnar nerve, with the exception of one fibular nerve. After treatment, 39 of 41 patients had functional recoveries or better. Conclusions: Electrodiagnosis can inform diagnosis of nerve injury and direct intervention including the need for surgical intervention. Electrodiagnosis should be considered for patients with initial signs of concomitant discrete nerve injury or with CRPS who are not responding to treatments because a nerve injury may be underlying.What is Known Complex Regional Pain Syndrome (CRPS) is a poorly understood pain condition. CRPS has been divided into two subtypes, the second subtype involves a discrete nerve injury with pain that extends beyond the territory of the nerve injury.What is New We observed that nerve injuries that may require surgical intervention are diagnosed just over half of the time upon initial assessment in patients with suspected CRPS. We observed that nerve injuries frequently required specifically directed interventions in place of or in conjunction with CRPS treatments. We suggest that electrodiagnosis is an important part of the triage protocol for CRPS II to reveal discrete nerve injuries that may be hidden. We recommend that electrodiagnosis be considered for patients with initial signs of concomitant discrete nerve injury or for CRPS patients who do not improve with medical therapies.

CRPS typically occurs after an injury or inciting event and can be accompanied by severe pain, sensitivity, swelling, changes in the color of the skin, changes in the texture of the skin, changes in hair and nail growth, weakness, muscle wasting, range of motion reduction, and temperature changes in the affected area. Little is known about type II CRPS, which may present with all of the same signs and symptoms as above but with damage to a nerve in the area. This study examined cases of CRPS where patients had electrodiagnostic findings that suggested a nerve injury. We found that the referring physicians reported nerve injuries in just over half of the cases. Oral prednisone was often the chosen treatment for these patients, although many required additional treatments, including surgery, to address their nerve injury. We recommend that electrodiagnosis be considered for patients with CRPS who are not responding to corticosteroids because a nerve injury could be preventing CRPS symptoms from resolving. When the CRPS trigger was a trauma that likely simultaneously injured the nerve, patients needed to have surgery more frequently than those with atraumatic onsets.

The Role of Fibrin(ogen) in Wound Healing and Infection Control.

Fibrinogen, one of the most abundant plasma proteins playing a key role in hemostasis, is an important modulator of wound healing and host defense against microbes. In the current review, we address the role of fibrin(ogen) throughout the process of wound healing and subsequent tissue repair. Initially fibrin(ogen) acts as a provisional matrix supporting incoming leukocytes and acting as reservoir for growth factors. It later goes on to support re-epithelialization, angiogenesis, and fibroplasia. Importantly, removal of fibrin(ogen) from the wound is essential for wound healing to progress. We also discuss how fibrin(ogen) functions through several mechanisms to protect the host against bacterial infection by providing a physical barrier, entrapment of bacteria in fibrin(ogen) networks, and by directing immune cell function. The central role of fibrin(ogen) in defense against bacterial infection has made it a target of bacterial proteins, evolved to interact with fibrin(ogen) to manipulate clot formation and degradation for the purpose of promoting microbial virulence and survival. Further understanding of the dual roles of fibrin(ogen) in wound healing and infection could provide novel means of therapy to improve recovery from surgical or chronic wounds and help to prevent infection from highly virulent bacterial strains, including those resistant to antibiotics.

High fibrinogen γ' levels in patient plasma increase clot formation at arterial and venous shear.

Fibrinogen γ' accounts for 3% to 40% of plasma fibrinogen. Earlier studies indicated that fibrinogen γ' forms altered fibrin clots under static conditions, whereas clinically, altered plasma γ' levels are associated with arterial and venous thrombosis. However, the effects of static vs flow conditions on the role of γ' throughout the pathophysiological range is unknown. This study explores the effects of γ' levels on clot formation and structure in static and flow conditions. Coagulation of plasma samples with low (n = 41; 3%), normal (n = 45; 10%), or high (n = 33; 30%) γ' levels were compared with that of purified fibrinogen mixtures with increasing ratios of γ' (3%, 10%, 30%). Clots were analyzed by confocal microscopy, permeation, turbidity, and lysis techniques. In a novel 2-step flow-perfusion model, fibrinogen-deficient plasma repleted with increasing ratios of γ' (3%, 10%, 30%) or plasmas with low (n = 5, 3%) or high (n = 5, 30%) γ' were flowed over preformed platelet aggregates at arterial (500 s-1) and venous (150 s-1) shear rates. Increasing γ' percentages within the pathophysiological range (3%-30%) did not result in any change in clot-formation rates; however, it led to significantly higher clot density, thinner fibers, and slower lysis in static conditions. Under flow at arterial shear, high γ' (30%) led to faster (+44.1%-75.3%) and increased (+104%-123%) fibrin deposition, with clots exhibiting a larger volume (+253%-655%) and height (+130%-146%). These trends were magnified at venous shear. Overall, our findings demonstrate the significant impact of pathophysiological fibrinogen γ' levels on clot structure and provide new flow-dependent mechanisms to explain how γ' increases thrombosis risk.

Thrombus Structural Composition in Cardiovascular Disease.

Thrombosis is a major complication of cardiovascular disease, leading to myocardial infarction, acute ischemic stroke, or venous thromboembolism. Thrombosis occurs when a thrombus forms inside blood vessels disrupting blood flow. Developments in thrombectomy to remove thrombi from vessels have provided new opportunities to study thrombus composition which may help to understand mechanisms of disease and underpin improvements in treatments. We aimed to review thrombus compositions, roles of components in thrombus formation and stability, and methods to investigate thrombi. Also, we summarize studies on thrombus structure obtained from cardiovascular patients and animal models. Thrombi are composed of fibrin, red blood cells, platelets, leukocytes, and neutrophil extracellular traps. These components have been analyzed by several techniques, including scanning electron microscopy, laser scanning confocal microscopy, histochemistry, and immunohistochemistry; however, each technique has advantages and limitations. Thrombi are heterogenous in composition, but overall, thrombi obtained from myocardial infarction are composed of mainly fibrin and other components, including platelets, red blood cells, leukocytes, and cholesterol crystals. Thrombi from patients with acute ischemic stroke are characterized by red blood cell- and platelet-rich regions. Thrombi from patients with venous thromboembolism contain mainly red blood cells and fibrin with some platelets and leukocytes. Thrombus composition from patients with myocardial infarction is influenced by ischemic time. Animal thrombosis models are crucial to gain further mechanistic information about thrombosis and thrombus structure, with thrombi being similar in composition compared with those from patients. Further studies on thrombus composition and function are key to improve treatment and clinical outcome of thrombosis.

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.

Recurrent venous thromboembolism patients form clots with lower elastic modulus than those formed by patients with non-recurrent disease.

Essentials Venous thromboembolism (VTE) recurrence leads to decreased clot elastic modulus in plasma. Recurrent VTE is not linked to changes in clot structure, fiber radius, or factor XIII activity. Other plasma components may play a role in VTE recurrence. Prospective studies should resolve if clot stiffness can be used as predictor for recurrent VTE. SUMMARY: Background Venous thromboembolism (VTE) is associated with a high risk of recurrent events after withdrawal of anticoagulation. Objectives To determine the difference in plasma clot mechanical properties between patients with recurrent VTE (rVTE) and those with non-recurrent VTE (nrVTE). Methods We previously developed a system for determining clot mechanical properties by use of an in-house magnetic tweezers system. This system was used to determine the mechanical properties of clots made from plasma of 11 patients with rVTE and 33 with nrVTE. Plasma was mixed with micrometer-sized beads, and thrombin and calcium were added to induce clotting; the mixture was then placed in small capillary tubes, and clotting was allowed to proceed overnight. Bead displacements upon manipulation with magnetic forces were analyzed to determine clot elastic and viscous moduli. Fibrin clot structure was analyzed with turbidimetry and confocal microscopy. Factor XIII was measured by pentylamine incorporation into fibrin. Results Clots from rVTE patients showed nearly two-fold less elastic and less viscous moduli than clots from nrVTE patients, regardless of male sex, unprovoked events, family history of VTE, fibrinogen concentration, or body mass index. No differences were observed in clot structure, fibrinolysis rates, or FXIII levels. Conclusion Using magnetic tweezers for the first time in patient samples, we found that plasma clots from rVTE patients showed a reduced elastic modulus and a reduced viscous modulus as compared with clots from nrVTE patients. These data indicate a possible role for fibrin clot viscoelastic properties in determining VTE recurrence.

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 because of its high cost and/or risk for infection. We present a novel methodology employing nonantibody 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 and confocal and electron microscopy. Binding studies and molecular modeling 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 minutes in the absence of Affimers to 172.6 ± 7.4 and more than 180 minutes (P < .0001), respectively, and from 7.6 ± 0.2 to 28.7 ± 5.8 (P < .05) and 149.3 ± 9.7 (P < .0001) minutes in clots made from purified fibrinogen. Prolongation in fibrinolysis was consistent across plasma samples from healthy control patients and individuals at high bleeding risk. F5 and G2 had a differential effect on clot structure and G2 profoundly altered fibrin fiber arrangement, whereas 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 1 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.

Assessment and determinants of whole blood and plasma fibrinolysis in patients with mild bleeding symptoms.

Enhanced clot lysis is associated with bleeding, but assessment of lysis capacity remains difficult. The plasma turbidity lysis and whole blood tissue Plasminogen Activator-Rotational Thromboelastometry (tPA-ROTEM) assays estimate fibrinolysis under more physiological conditions than clinically used assays. We hypothesized that these assays could find signs of enhanced lysis capacity in patients who report bleeding symptoms, but are not diagnosed with bleeding disorders. We also aimed to gain insight in determinants of the results of these lysis assays. Data from 240 patients with and 95 patients without self-reported bleeding symptoms were obtained, who were included in a study that primarily aimed to assess prevalence of haemostatic abnormalities in preoperative patients. ROTEM and turbidity assays were performed with rtPA. Blood counts, fibrinolysis and coagulation factor activities were determined. Data were analysed using multivariable linear regression models. Remarkably, patients reporting bleeding symptoms showed signs of significantly impaired lysis capacity in the tPA-ROTEM, but not in the turbidity lysis assay. In these patients, the tPA-ROTEM results depended on FII, FXII, plasminogen, α2-antiplasmin, PAI-1 and TAFI levels. The turbidity lysis results were significantly influenced by fibrinogen, α2-antiplasmin, PAI-1 and TAFI. In conclusion, the tPA-ROTEM and the turbidity lysis assay could not detect enhanced fibrinolytic capacity in patients with bleeding symptoms. This suggests that these symptoms are not caused by enhanced fibrinolytic activity. As both assays were sensitive to important determinants of fibrinolysis they may be able to detect a fibrinolytic imbalance, but this needs to be validated in patients with known hypo- or hyperfibrinolytic disorders.

Impact of γ'γ' fibrinogen interaction with red blood cells on fibrin clots.

AIM: γ' fibrinogen has been associated with thrombosis. Here the interactions between γ'γ' or γAγA fibrinogen and red blood cells (RBCs), and their role on fibrin clot properties were studied. MATERIALS & METHODS: Atomic Force microscopy (AFM)-based force spectroscopy, rheological, electron and confocal microscopy, and computational approaches were conducted for both fibrinogen variants. RESULTS & CONCLUSION: AFM shows that the recombinant human (rh)γ'γ' fibrinogen increases the binding force and the frequency of the binding to RBCs compared with rhγAγA, promoting cell aggregation. Structural changes in rhγ'γ' fibrin clots, displaying a nonuniform fibrin network were shown by microscopy approaches. The presence of RBCs decreases the fibrinolysis rate and increases viscosity of rhγ'γ' fibrin clots. The full length of the γ' chain structure, revealed by computational analysis, occupies a much wider surface and is more flexible, allowing an increase of the binding between γ' fibers, and eventually with RBCs.

A fibrin biofilm covers blood clots and protects from microbial invasion.

Hemostasis requires conversion of fibrinogen to fibrin fibers that generate a characteristic network, interact with blood cells, and initiate tissue repair. The fibrin network is porous and highly permeable, but the spatial arrangement of the external clot face is unknown. Here we show that fibrin transitioned to the blood-air interface through Langmuir film formation, producing a protective film confining clots in human and mouse models. We demonstrated that only fibrin is required for formation of the film, and that it occurred in vitro and in vivo. The fibrin film connected to the underlying clot network through tethering fibers. It was digested by plasmin, and formation of the film was prevented with surfactants. Functionally, the film retained blood cells and protected against penetration by bacterial pathogens in a murine model of dermal infection. Our data show a remarkable aspect of blood clotting in which fibrin forms a protective film covering the external surface of the clot, defending the organism against microbial invasion.

The 95RGD97 sequence on the Aα 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 ß3 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.