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Blood-brain barrier disruption, microglial activation and neurodegeneration are hallmarks of multiple sclerosis. However, the initial triggers that activate innate immune responses and their role in axonal damage remain unknown. Here we show that the blood protein fibrinogen induces rapid microglial responses toward the vasculature and is required for axonal damage in neuroinflammation. Using in vivo two-photon microscopy, we demonstrate that microglia form perivascular clusters before myelin loss or paralysis onset and that, of the plasma proteins, fibrinogen specifically induces rapid and sustained microglial responses in vivo . Fibrinogen leakage correlates with areas of axonal damage and induces reactive oxygen species release in microglia. Blocking fibrin formation with anticoagulant treatment or genetically eliminating the fibrinogen binding motif recognized by the microglial integrin receptor CD11b/CD18 inhibits perivascular microglial clustering and axonal damage. Thus, early and progressive perivascular microglial clustering triggered by fibrinogen leakage upon blood-brain barrier disruption contributes to axonal damage in neuroinflammatory disease. Multiple sclerosis is characterized by the activation of microglia cells. Davalos et al . investigate the early stages of neuroinflammation in mice and reveal that the plasma protein fibrinogen induces microglial clustering around the brain vasculature, which facilitates lesion formation and focal axonal damage.

Blood platelets aggregate to form clots that prevent haemorrhage. Knowledge of single-platelet mechanics is scarce, however. Atomic force microscopy experiments now show that platelets contract rapidly on contact with fibrinogen, and adhere strongly to multiple fibrin polymers, enhancing the elasticity of clots. These findings are relevant to disorders of platelet function, such as thrombosis. Platelets interact with fibrin polymers to form blood clots at sites of vascular injury 1 , 2 , 3 . Bulk studies have shown clots to be active materials, with platelet contraction driving the retraction and stiffening of clots 4 . However, neither the dynamics of single-platelet contraction nor the strength and elasticity of individual platelets, both of which are important for understanding clot material properties, have been directly measured. Here we use atomic force microscopy to measure the mechanics and dynamics of single platelets. We find that platelets contract nearly instantaneously when activated by contact with fibrinogen and complete contraction within 15 min. Individual platelets can generate an average maximum contractile force of 29 nN and form adhesions stronger than 70 nN. Our measurements show that when exposed to stiffer microenvironments, platelets generated higher stall forces, which indicates that platelets may be able to contract heterogeneous clots more uniformly. The high elasticity of individual platelets, measured to be 10 kPa after contraction, combined with their high contractile forces, indicates that clots may be stiffened through direct reinforcement by platelets as well as by strain stiffening of fibrin under tension due to platelet contraction. These results show how the mechanosensitivity and mechanics of single cells can be used to dynamically alter the material properties of physiologic systems.

Background Hidden blood loss (HBL) represents an important complication of unilateral biportal endoscopic (UBE) spine surgery. This study aimed to evaluate HBL and its possible risk factors among patients undergoing UBE surgery for lumbar degenerative diseases. Methods This multicentric retrospective study was conducted in 3 different medical centers between July 2020 and April 2021. Data of patients who underwent UBE surgery were extracted by electronic medical record system. The patient’s demographic characteristics and blood loss-related parameters were recorded. We calculated the amount of HBL and explored the association between patient’s characteristics and HBL using Pearson or Spearman correlation analysis. Multivariate linear regression analysis was conducted to identify independent risk factors of HBL. Results A total of 136 patients (55 females and 81 males, age range 43 to 74 years) were included in this study. A substantial amount of HBL (469.5 ± 195.3 ml, 57.6% of TBL, total blood loss) occurred following UBE surgery. Multiple linear regression analysis indicated that the risk factors of HBL were as follows: age ( P = 0.000), number of fusion levels ( P = 0.015), American Society of Anesthesiologists (ASA) classification ( P = 0.046), surgery time ( P = 0.017), patient’s blood volume (PBV, P = 0.026), total blood loss (TBL, P = 0.001), postoperative (i.e., day 2 or 3) hematocrit (Hct, P = 0.034), Hct loss ( P = 0.005), and fibrinogen ( P = 0.028). Conclusions A certain amount of HBL occurs in UBE surgery and cannot be ignored in daily clinical practice. The age, number of fusion levels, ASA classification, surgery time, PBV, TBL, postoperative Hct, Hct loss, and fibrinogen are independent risk factors for HBL.

Non-genetic MMP-2 insufficiency is a relatively unexplored condition which could be induced by pathological overexpression of endogenous MMP-2 inhibitors such as TIMPs and/or the acute phase reactant alpha-2-macroglobulin. Here, we investigate the hypothesis that human fibrinogen (FBG) – an acute phase reactant – inhibits human MMP-2. Following an unexpected observation where sera from human donors including arthritis patients with increased levels of serum FBG exhibited reduced binding of serum proMMP-2 to gelatin, we found that human FBG (0 to 3.6 mg/mL i.e., 0 to 10.6 μM) concentration-dependently inhibited human proMMP-2 and MMP2 from binding to gelatin. Moreover, at normal physiological concentrations, FBG (5.29–11.8 μM) concentration-dependently inhibited (40–70% inhibition) the cleavage of fluorescein-conjugated gelatin by MMP-2, but not MMP-9. Indicative of a mixed-type (combination of competitive and non-competitive) inhibition mechanism, FBG reduced the V max (24.9 ± 0.7 min −1 to 17.7 ± 0.9 min −1 , P < 0.05) and increased the Michaelis-Menten constant K M (204 ± 6 n M to 478 ± 50 nM, P < 0.05) for the reaction of MMP-2 cleavage of fluorescein-conjugated gelatin. In silico analyses and studies of FBG neutralization with anti-FBG antibodies implicated the domains D and E of FBG in the inhibition of MMP-2. In conclusion, FBG is a natural selective MMP-2 inhibitor, whose pathological elevation could lead to MMP-2 insufficiency in humans.

We investigated the relationship between fibrinolytic factors and computed tomography (CT) findings in patients with chronic subdural hematomas (CSDHs). Thirty-one patients with CSDHs were divided on the basis of CT findings into heterogeneous and homogeneous groups. A sample from the subdural hematoma was obtained at surgery to measure the concentrations of fibrinogen and D-dimer. The mean level of fibrinogen in the heterogeneous group, including the layering ( n = 4) and mixed ( n = 10) type, was 88.2 ± 121.2 mg/dL, whereas in the homogeneous group, including high density ( n = 2), isodensity ( n = 9), and low density ( n = 6) types, it was <25 mg/dL. The concentration of fibrinogen was significantly higher in the heterogeneous group than in the homogeneous group ( p = 0.006). The mean level of D-dimer in the heterogeneous group was 35,407.9 ± 16,325.5 μg/L, whereas for the homogeneous group it was 1476.4 ± 2091.4 μg/L. The concentration of D-dimer was significantly higher in the heterogeneous group than in the homogeneous group ( p < 0.001). The layering and mixed types of CSDH exhibited higher concentrations of fibrinogen and D-dimer in subdural hematoma than the homogeneous types. These fibrinolytic factors appear to be associated with evolution in CSDHs with heterogeneous density.

Changes in the molecular and cellular composition of the CNS after injury or disease result in the formation of an inhibitory environment that inhibits the regeneration of adult mammalian CNS neurons. Although a dramatic change in the CNS environment after traumatic injury or disease is hemorrhage because of vascular rupture or leakage of the blood-brain barrier, the potential role for blood proteins in repair processes remains unknown. Here we show that the blood protein fibrinogen is an inhibitor of neurite outgrowth that is massively deposited in the spinal cord after injury. We show that fibrinogen acts as a ligand for beta3 integrin and induces the transactivation of EGF receptor (EGFR) in neurons. Fibrinogen-mediated inhibition of neurite outgrowth is reversed by blocking either beta3 integrin or phoshorylation of EGFR. Inhibition of Src family kinases that mediate the cross-talk between integrin and growth factor receptors rescue the fibrinogen-induced phosphorylation of EGFR. These results identify fibrinogen as the first blood-derived inhibitor of neurite outgrowth and suggest fibrinogen-induced EGFR transactivation on neuronal cells as a molecular link between vascular and neuronal damage in the CNS after injury.

The binding of bacteria to fibrinogen and platelets are important events in the pathogenesis of infective endocarditis. Srr1 is a serine-rich repeat glycoprotein of Streptococcus agalactiae that binds directly to the Aα chain of human fibrinogen. To assess the impact of Srr1 on the pathogenesis of endocarditis due to S. agalactiae, we first examined the binding of this organism to immobilized human platelets. Strains expressing Srr1 had significantly higher levels of binding to human platelets in vitro, as compared with isogenic Δsrr1 mutants. In addition, platelet binding was inhibited by pretreatment with anti-fibrinogen IgG or purified Srr1 binding region. To assess the contribution of Srr1 to pathogenicity, we compared the relative virulence of S. agalactiae NCTC 10/84 strain and its Δsrr1 mutant in a rat model of endocarditis, where animals were co-infected with the WT and the mutant strains at a 1:1 ratio. At 72 h post-infection, bacterial densities (CFU/g) of the WT strain within vegetations, kidneys, and spleens were significantly higher, as compared with the Δsrr1 mutant. These results indicate that Srr1 contributes to the pathogenesis of endocarditis due to S. agalactiae, at least in part through its role in fibrinogen-mediated platelet binding.

Staphylococcus aureus is a prominent bacterial pathogen that is known to agglutinate in the presence of human plasma to form stable clumps. There is increasing evidence that agglutination aids S. aureus pathogenesis, but the mechanisms of this process remain to be fully elucidated. To better define this process, we developed both tube based and flow cytometry methods to monitor clumping in the presence of extracellular matrix proteins. We discovered that the ArlRS two-component system regulates the agglutination mechanism during exposure to human plasma or fibrinogen. Using divergent S. aureus strains, we demonstrated that arlRS mutants are unable to agglutinate, and this phenotype can be complemented. We found that the ebh gene, encoding the Giant Staphylococcal Surface Protein (GSSP), was up-regulated in an arlRS mutant. By introducing an ebh complete deletion into an arlRS mutant, agglutination was restored. To assess whether GSSP is the primary effector, a constitutive promoter was inserted upstream of the ebh gene on the chromosome in a wildtype strain, which prevented clump formation and demonstrated that GSSP has a negative impact on the agglutination mechanism. Due to the parallels of agglutination with infective endocarditis development, we assessed the phenotype of an arlRS mutant in a rabbit combined model of sepsis and endocarditis. In this model the arlRS mutant displayed a large defect in vegetation formation and pathogenesis, and this phenotype was partially restored by removing GSSP. Altogether, we have discovered that the ArlRS system controls a novel mechanism through which S. aureus regulates agglutination and pathogenesis.

Engineered biomatrices offer the potential to recapitulate the regenerative microenvironment, with important implications in tissue repair. In this context, investigation of the molecular interactions occurring between growth factors, cytokines and extracellular matrix (ECM) has gained increasing interest. Here, we sought to investigate the possible interactions between the ECM proteins fibronectin (FN) and fibrinogen (Fg) with the CXCR3 ligands CXCL9, CXCL10 and CXCL11, which are expressed during wound healing. New binding interactions were observed and characterized. Heparin-binding domains within Fg (residues 15-66 of the β chain, Fg β15-66) and FN (FNI1-5, but not FNIII12-14) were involved in binding to CXCL10 and CXCL11 but not CXCL9. To investigate a possible influence of FN and Fg interactions with CXCL11 in mediating its role during re-epithelialization, we investigated human keratinocyte migration in vitro and wound healing in vivo in diabetic db/db mice. A synergistic effect on CXCL11-induced keratinocyte migration was observed when cells were treated with CXCL11 in combination with FN in a transmigration assay. Moreover, wound healing was enhanced in full thickness excisional wounds treated with fibrin matrices functionalized with FN and containing CXCL11. These findings highlight the importance of the interactions occurring between cytokines and ECM and point to design concepts to develop functional matrices for regenerative medicine.

The objective of this study is to determine how a hibernating mammal avoids the formation of blood clots under periods of low blood flow. A microfluidic vascular injury model was performed to differentiate the effects of temperature and shear rate on platelet adhesion to collagen. Human and ground squirrel whole blood was incubated at 15 or 37 °C and then passed through a microfluidic chamber over a 250-µm strip of type I fibrillar collagen at that temperature and the shear rates of 50 or 300 s −1 to simulate torpid and aroused conditions, respectively. At 15 °C, both human and ground squirrel platelets showed a 90–95% decrease in accumulation on collagen independent of shear rate. At 37 °C, human platelet accumulation reduced by 50% at 50 s −1 compared to 300 s −1 , while ground squirrel platelet accumulation dropped by 80%. When compared to platelets from non-hibernating animals, platelets from animals collected after arousal from torpor showed a 60% decrease in binding at 37 °C and 300 s −1 , but a 2.5-fold increase in binding at 15 °C and 50 s −1 . vWF binding in platelets from hibernating ground squirrels was decreased by 50% relative to non-hibernating platelets. The source of the plasma that platelets were stored in did not affect the results indicating that the decreased vWF binding was a property of the platelets. Upon chilling, ground squirrel platelets increase microtubule assembly leading to the formation of long rods. This shape change is concurrent with sequestration of platelets in the liver and not the spleen. In conclusion, it appears that ground squirrel platelets are sequestered in the liver during torpor and have reduced binding capacity for plasma vWF and lower accumulation on collagen at low shear rates and after storage at cold temperatures, while still being activated by external agonists. These adaptations would protect the animals from spontaneous thrombus formation during torpor but allow them to restore normal platelet function upon arousal.