The role of fibrinogen in traumatic brain injury: from molecular pathological mechanisms to clinical management.

Fibrinogen is the substrate of plasma coagulation. It plays an important role in the formation of reticular network, which is crucial to the strength and stability of blood clots. In addition to directly participating in coagulation, fibrinogen also participates in the destruction of blood-brain barrier and neuroinflammation. This article reviews the pathophysiological changes of fibrinogen after traumatic brain injury. Considerable efforts have been made to understand the mechanisms by which fibrinogen damages the central nervous system. Combined with the latest research hotspots, potentially promising treatment strategies at the molecular level were discussed. We believe that understanding the role of fibrinogen-mediated damage in nerve and blood-brain barrier function will enable timely intervention in patients with nerve damage, and guide the development of novel targeted therapeutics.

Analysis of risk factors for perioperative hidden blood loss in unilateral biportal endoscopic spine surgery: a retrospective multicenter study.

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.

Fibrinogen-Like Protein 1 Modulates Sorafenib Resistance in Human Hepatocellular Carcinoma Cells.

Despite liver cancer being the second-leading cause of cancer-related death worldwide, few systemic drugs have been approved. Sorafenib, the first FDA-approved systemic drug for unresectable hepatocellular carcinoma (HCC), is limited by resistance. However, the precise mechanisms underlying this phenomenon are unknown. Since fibrinogen-like 1 (FGL1) is involved in HCC progression and upregulated after anticancer therapy, we investigated its role in regulating sorafenib resistance in HCC. FGL1 expression was assessed in six HCC cell lines (HepG2, Huh7, Hep3B, SNU387, SNU449, and SNU475) using western blotting. Correlations between FGL1 expression and sorafenib resistance were examined by cell viability, colony formation, and flow cytometry assays. FGL1 was knocked-down to confirm its effects on sorafenib resistance. FGL1 expression was higher in HepG2, Huh7, and Hep3B cells than in SNU387, SNU449, and SNU475 cells; high FGL1-expressing HCC cells showed a lower IC50 and higher sensitivity to sorafenib. In Huh7 and Hep3B cells, FGL1 knockdown significantly increased colony formation by 61% (p = 0.0013) and 99% (p = 0.0002), respectively, compared to that in controls and abolished sorafenib-induced suppression of colony formation, possibly by modulating ERK and autophagy signals. Our findings demonstrate that sorafenib resistance mediated by FGL1 in HCC cells, suggesting FGL1 as a potential sorafenib-resistance biomarker and target for HCC therapy.

FGL2-wired macrophages secrete CXCL7 to regulate the stem-like functionality of glioma cells.

Glioma stem cells (GSCs) are thought to underlie glioma initiation, evolution, resistance to therapies, and relapse. They are defined by their capacity to initiate glioma in immunocompromised mice which precludes analysis of their interaction with immune cells. Macrophages dominate the immune cell composition in glioma. We hypothesized that stemness and immune evasion induced by macrophages are closed intertwined in glioma. By using mass cytometry and RNA sequencing, we reveal that in immunocompetent mice, FGL2 promotes the stem-like phenotypes of glioma cells in an expression level-dependent manner. Mechanistically, FGL2-producing glioma cells recruit macrophages into the tumor microenvironment and induce the macrophages to secrete CXCL7 via the CD16/SyK/PI3K/HIF1α pathways. CXCL7, in turn, enhances the stem-like functionality of glioma cells, resulting in an increase in tumor incidence and progression that can be blocked with a neutralizing anti-CXCL7 antibody. Clinically, the FGL2-CXCL7 paracrine loop positively correlated with a higher macrophage signature and poorer prognosis in glioma patients. Thus, glioma cells' stem-like functionality is regulated by FGL2 in the presence of macrophages, and the FGL2-CXCL7 paracrine signaling axis is critical for regulating this function.

Protamine stimulates platelet aggregation in vitro with activation of the fibrinogen receptor and alpha-granule release, but impairs secondary activation via ADP and thrombin receptors.

Heparin and protamine are fundamental in the management of anticoagulation during cardiac surgery. Excess protamine has been associated with increased bleeding. Interaction between protamine and platelet function has been demonstrated but the mechanism remains unclear. We examined the effect of protamine on platelet function in vitro using impedance aggregometry, flow cytometry, and thrombin generation. Platelets were exposed to protamine at final concentrations of 0, 20, 40, and 80 µg/mL, alone or together with adenosine diphosphate (ADP) or thrombin PAR1 receptor-activating peptide (TRAP). We found that in the absence of other activators, protamine (80 µg/mL) increased the proportion of platelets with active fibrinogen receptor (binding of PAC-1) from 3.6% to 97.0% (p < .001) measured with flow cytometry. Impedance aggregometry also increased slightly after exposure to protamine alone. When activated with ADP or TRAP protamine at 80 µg/mL reduced aggregation, from 73.8 ± 29.4 U to 46.9 ± 21.1 U (p < .001) with ADP and from 126.4 ± 16.1 U to 94.9 ± 23.7 U (p < .01) with TRAP. P-selectin exposure (a marker of alpha-granule release) measured by median fluorescence intensity (MFI) increased dose dependently with protamine alone, from 0.76 ± 0.20 (0 µg/mL) to 10.2 ± 3.1 (80 µg/mL), p < .001. Protamine 80 µg/mL by itself resulted in higher MFI (10.16 ± 3.09) than activation with ADP (2.2 ± 0.7, p < .001) or TRAP (5.7 ± 2.6, p < .01) without protamine. When protamine was combined with ADP or TRAP, there was a concentration-dependent increase in the alpha-granule release. In conclusion, protamine interacts with platelets in vitro having both a direct activating effect and impairment of secondary activation of aggregation by other agonists.

COVID-19 Associated Hypercoagulability: Manifestations, Mechanisms, and Management.

ABSTRACT: Patients with severe coronavirus disease-2019 (COVID-19) frequently have hypercoagulability caused by the immune response to the severe acute respiratory syndrome coronavirus-2 infection. The pathophysiology of COVID-19 associated hypercoagulability is not fully understood, but characteristic changes include: increased fibrinogen concentration, increased Factor VIII activity, increased circulating von Willebrand factor, and exhausted fibrinolysis. Anticoagulant therapy improves outcomes in mechanically ventilated patients with COVID-19 and viscoelastic coagulation testing offers an opportunity to tailor anticoagulant therapy based on an individual patient's coagulation status. In this narrative review, we summarize clinical manifestations of COVID-19, mechanisms, monitoring considerations, and anticoagulant therapy. We also review unique considerations for COVID-19 patients who are on extracorporeal membrane oxygenation.

Fibrinogen Activates PAK1/Cofilin Signaling Pathway to Protect Endothelial Barrier Integrity.

INTRODUCTION: We recently demonstrated that fibrinogen stabilizes syndecan-1 on the endothelial cell (EC) surface and contributes to EC barrier protection, though the intracellular signaling pathway remains unclear. P21 (Rac1) activated kinase 1 (PAK1) is a protein kinase involved in intracellular signaling leading to actin cytoskeleton rearrangement and plays an important role in maintaining endothelial barrier integrity. We therefore hypothesized that fibrinogen binding to syndecan-1 activated the PAK1 pathway. METHODS: Primary human lung microvascular endothelial cells were incubated in 10% lactated Ringers (LR) solution or 10% fibrinogen saline solution (5 mg/mL). Protein phosphorylation was determined by Western blot analysis and endothelial permeability measured by fluorescein isothiocyanate (FITC)-dextran. Cells were silenced by siRNA transfection. Protein concentration was measured in the lung lavages of mice. RESULTS: Fibrinogen treatment resulted in increased syndecan-1, PAK1 activation (phosphorylation), cofilin activation (dephosphorylation), as well as decreased stress fibers and permeability when compared with LR treatment. Cofilin is an actin-binding protein that depolymerizes F-actin to decrease stress fiber formation. Notably, fibrinogen did not influence myosin light chain activation (phosphorylation), a mediator of EC tension. Silencing of PAK1 prevented fibrinogen-induced dephosphorylation of cofilin and barrier integrity. Moreover, to confirm the in vitro findings, mice underwent hemorrhagic shock and were resuscitated with either LR or fibrinogen. Hemorrhage shock decreased lung p-PAK1 levels and caused significant lung vascular leakage. However, fibrinogen administration increased p-PAK1 expression to near sham levels and remarkably prevented the lung leakage. CONCLUSION: We have identified a novel pathway by which fibrinogen activates PAK1 signaling to stimulate/dephosphorylate cofilin, leading to disassembly of stress fibers and reduction of endothelial permeability.

Basic principles of viscoelastic testing.

BACKGROUND: Viscoelastic testing is a method of hemostatic analysis that provides a real-time, holistic view of ex vivo clotting. It allows for examination of both cellular and plasma protein contributions to clotting including platelet number and function, fibrin(ogen) function, and coagulation factor function. The method assesses physical clot properties during the transition of blood from a liquid to a gel state, either by measurement of clot shear modulus using physical force transduction or by measurement of clot resonance frequency using sonometric interrogation. Results are reported in a live trace, with different trace parameters reflecting different contributors to hemostasis. These reported parameters vary between testing platforms. RESULTS: In the United States, there are several commonly used Food and Drug Administration (FDA)-approved viscoelastic instruments available on the market. Those instruments that use sonometric clot assessment are more recently available and allow for improved portability for use near the patient's bedside. These instruments generally feature different reagent kits that allow more specific interrogation of different hemostatic pathways. Viscoelastic testing can predict the results of traditional plasma-based coagulation assays and has the added benefit of detecting hypercoagulability and severe hyperfibrinolysis. Implementation of viscoelastic testing in many clinical settings is becoming widespread and has proven to be efficacious in reducing blood transfusion rates in many settings. An impact on overall mortality and morbidity has not yet been demonstrated. CONCLUSION: This article provides a narrative review of the basic principles of viscoelastic testing, including the science and technology behind the method, as well as currently available testing platforms and reagents.

The protective rather than prothrombotic fibrinogen in COVID-19 and other inflammatory states.

Hypercoagulability has been recognized as a common complication of COVID-19. Exact mechanisms for this extreme coagulation activation have not yet been elucidated. However, one of the consistent laboratory finding is the increase in fibrinogen, in some cases, marked elevation. High circulating levels of fibrinogen have been linked to thrombosis for years and for this reason, hyperfibrinogenemia is considered one of the mechanisms for COVID-19 coagulopathy. In this forum article, instead of the prothrombotic role, a protective function for fibrinogen is discussed. Fibrinogen, like the other well-known acute phase reactants, is increased in COVID-19 possibly to protect the host.

Comparison of Neonatal and Adult Fibrin Clot Properties between Porcine and Human Plasma.

BACKGROUND: Recent studies suggest that adult-specific treatment options for fibrinogen replacement during bleeding may be less effective in neonates. This is likely due to structural and functional differences found in the fibrin network between adults and neonates. In this investigation, the authors performed a comparative laboratory-based study between immature and adult human and porcine plasma samples in order to determine if piglets are an appropriate animal model of neonatal coagulopathy. METHODS: Adult and neonatal human and porcine plasma samples were collected from the Children's Hospital of Atlanta and North Carolina State University College of Veterinary Medicine, respectively. Clots were formed for analysis and fibrinogen concentration was quantified. Structure was examined through confocal microscopy and cryogenic scanning electron microscopy. Function was assessed through atomic force microscopy nanoindentation and clotting and fibrinolysis assays. Lastly, novel hemostatic therapies were applied to neonatal porcine samples to simulate treatment. RESULTS: All sample groups had similar plasma fibrinogen concentrations. Neonatal porcine and human plasma clots were less branched with lower fiber densities than the dense and highly branched networks seen in adult human and porcine clots. Neonatal porcine and human clots had faster degradation rates and lower clot stiffness values than adult clots (stiffness [mmHg] mean ± SD: neonatal human, 12.15 ± 1.35 mmHg vs. adult human, 32.25 ± 7.13 mmHg; P = 0.016; neonatal pig, 10.5 ± 8.25 mmHg vs. adult pigs, 32.55 ± 7.20 mmHg; P = 0.015). The addition of hemostatic therapies to neonatal porcine samples enhanced clot formation. CONCLUSIONS: The authors identified similar age-related patterns in structure, mechanical, and degradation properties between adults and neonates in porcine and human samples. These findings suggest that piglets are an appropriate preclinical model of neonatal coagulopathy. The authors also show the feasibility of in vitro model application through analysis of novel hemostatic therapies as applied to dilute neonatal porcine plasma.

Role of placental fibrinogen-like protein 1 in gestational diabetes.

In view of the increasing prevalence of gestational diabetes mellitus (GDM) and the increased risks of delivering a macrosomic infant, developing preeclampsia, and suffering a perinatal death due to GDM, GDM has emerged as a growing public health problem. Although the placenta was suggested to play a crucial role in the pathology of GDM, the mechanisms that induce the development of GDM are still obscure. Fibrinogen-like protein (FGL)-1 is a hepatokine that plays an important role in hepatogenesis, as well as in nonalcoholic fatty liver disease and diabetes. Although FGL-1 is also expressed by the placenta, the pathophysiological role of FGL-1 in GDM is still unknown. In this study, FGL-1 levels were evaluated in 45 subjects with (n = 16) or without (n = 29) GDM. We found that FGL-1 was mainly expressed by placental trophoblasts, and FGL-1 expression was significantly higher in subjects with GDM. FGL-1 increased trophoblast proliferation through an extracellular signal-regulated kinase 1/2-dependent pathway. In addition, plasma concentrations of FGL-1 were higher in subjects with GDM, and the increased circulating FGL-1 might contribute to systemic insulin resistance. FGL-1 disrupted the gluconeogenic action of insulin in HepG2 cells, and decreased insulin-induced glucose uptake by L6 myotubes. Taken together, placental FGL-1 possibly plays a role in the impairment of insulin function in the development of GDM, and it might be a novel biomarker for diagnosing GDM.

Clotting factors: Clinical biochemistry and their roles as plasma enzymes.

The purpose of this review is to describe structure and function of the multiple proteins of the coagulation system and their subcomponent domains. Coagulation is the process by which flowing liquid blood plasma is converted to a soft, viscous gel entrapping the cellular components of blood including red cells and platelets and thereby preventing extravasation of blood. This process is triggered by the minimal proteolysis of plasma fibrinogen. This transforms the latter to sticky fibrin monomers which polymerize into a network. The proteolysis of fibrinogen is a function of the trypsin-like enzyme termed thrombin. Thrombin in turn is activated by a cascade of trypsin-like enzymes that we term coagulation factors. In this review we examine the mechanics of the coagulation cascade with a view to the structure-function relationships of the proteins. We also note that two of the factors have no trypsin like protease domain but are essential cofactors or catalysts for the proteases. This review does not discuss the major role of platelets except to highlight their membrane function with respect to the factors. Coagulation testing is a major part of routine diagnostic clinical pathology. Testing is performed on specimens from individuals either with bleeding or with thrombotic disorders and those on anticoagulant medications. We examine the basic in-vitro laboratory coagulation tests and review the literature comparing the in vitro and in vivo processes. In vitro clinical testing typically utilizes plasma specimens and non-physiological or supraphysiological activators. Because the review focuses on coagulation factor structure, a brief overview of the evolutionary origins of the coagulation system is included.

Identification of Fibrinogen as a Key Anti-Apoptotic Factor in Human Fresh Frozen Plasma for Protecting Endothelial Cells In Vitro.

Resuscitation with human fresh frozen plasma (FFP) in hemorrhagic shock (HS) patients is associated with improved clinical outcomes. Our group has demonstrated that the beneficial effect of FFP is due to its blockade on endothelial hyperpermeability, thereby improving vascular barrier function. The current study aimed to investigate HS-induced endothelial cell apoptosis, a potential major contributor to the endothelial hyperpermeability, and to determine the effect and the key components/factors of FFP on protecting endothelial cells from apoptosis. We first measured and demonstrated an increase in apoptotic endothelial microparticles (CD146AnnexinV) in patients in shock compared to normal subjects, indicating the induction of endothelial cell activation and apoptosis in shock patients. We then transfused HS rats with FFP and showed that FFP blocked HS-induced endothelial cell apoptosis in gut tissue. To identify the anti-apoptotic factors in FFP, we utilized high-performance liquid chromatography, fractionated FFP, and screened the fractions in vitro for the anti-apoptotic effects. We selected the most effective fractions, performed mass spectrometry, and identified fibrinogen as a potent anti-apoptotic factor. Taken together, our findings suggest that HS-induced endothelial apoptosis may constitute a major mechanism underlying the vascular hyperpermeability. Furthermore, the identified anti-apoptotic factor fibrinogen may contribute to the beneficial effects of FFP resuscitation, and therefore, may have therapeutic potential for HS.

MONITORING OF HEMORHEOLOGICAL PARAMETERS IN PATIENTS WITH ATRIAL FIBRILLATION (INITIAL DATA).

The goals of this project were to determine how hemodynamics and hemorheological change in patients diagnosed with different forms of atrial fibrillation; also, how relievers of changes in fibrinogen monitoring in patients with permanent, persistent, paroxysmal forms of arrhythmias. There was examined 30 patients (the average age of patients 65). Patients and control groups have been tested the following studies: index of erythrocytes aggregability, deformability, plasma viscosity to evaluate the blood rheological parameter, also -Fibrinogen to determine of coagulation condition. The Index of the Erythrocytes aggregation was done with the system of textural analyzes. These new innovative methods "Georgian Technique" is created by Georgian scientists and they are famous in the world as direct, numeral and exact. The index of the deformation of the erythrocytes was done with filtration method. Plasma viscosity was measured in the capillary viscosimeter in 370 C. According to the obtained data patients with atrial fibrillation have the same conditions of rheological and coagulation systems, despite of the forms of the atrial fibrillation. And it is different compared to the control group. In the statistical processing of the total row of fibrinogen, the patients were divided into 3 categories. As it turned out fibrinogen and Index of erythrocytes aggregation are in a linear relationship. The quantity of fibrinogen and of erythrocytes aggregation increase simultaneously and the greater the sequential number of the category is, the changes are more pronounced. However, the change/variability of each biological parameter, as shown from our data, is not uniform and linear. The obtained results clearly illustrate the existence of two parallel mechanisms in the body. These are on the one hand the systems of coagulation, anti-coagulation and fibtinollysis. These processes are in a state of the weighted condition, and they are characterized by dynamic equilibrium. On the other hand, the rheology system, which involves the combination of blood flow, blood velocity, vascular stiffness / elasticity, is characterized by one direction going on, with the adequacy and non-existing antipodal mechanisms. The hemorheological system does not have a physiologically balanced opposing anti-rheological system. All of this generate that the hemorheological status is very important in the development and formation of some disease. The arrangement of a rheologycal system is taking preventive character. On the one hand, the rheologycal system is a consolidation of diagnosis and evaluation of the mechanisms. Also, it is the treatment target. Normalization of them is very important in the therapeutic standpoint of the individual.

Magnetic Resonance Imaging Reveals Distinct Roles for Tissue Transglutaminase and Factor XIII in Maternal Angiogenesis During Early Mouse Pregnancy.

OBJECTIVE: The early embryo implantation is characterized by enhanced uterine vascular permeability at the site of blastocyst attachment, followed by extracellular-matrix remodeling and angiogenesis. Two TG (transglutaminase) isoenzymes, TG2 (tissue TG) and FXIII (factor XIII), catalyze covalent cross-linking of the extracellular-matrix. However, their specific role during embryo implantation is not fully understood. Approach and Results: For mapping the distribution as well as the enzymatic activities of TG2 and FXIII towards blood-borne and resident extracellular-matrix substrates, we synthetized selective and specific low molecular weight substrate analogs for each of the isoenzymes. The implantation sites were challenged by genetically modifying the trophoblast cells in the outer layer of blastocysts, to either overexpress or deplete TG2 or FXIII, and the angiogenic response was studied by dynamic contrast-enhanced-magnetic resonance imaging. Dynamic contrast-enhanced-magnetic resonance imaging revealed a decrease in the permeability of decidual vasculature surrounding embryos in which FXIII were overexpressed in trophoblast cell. Reduction in decidual blood volume fraction was demonstrated when either FXIII or TG2 were overexpressed in embryonic trophoblast cell and was elevated when trophoblast cell was depleted of FXIII. These results were corroborated by histological analysis. CONCLUSIONS: In this study, we report on the isoenzyme-specific roles of TG2 and FXIII during the early days of mouse pregnancy and further reveal their involvement in decidual angiogenesis. Our results reveal an important magnetic resonance imaging-detectable function of embryo-derived TG2 and FXIII on regulating maternal angiogenesis during embryo implantation in mice.Visual Overview: An online visual overview is available for this article.

Identification of fibrinogen as a natural inhibitor of MMP-2.

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 Vmax (24.9 ± 0.7 min-1 to 17.7 ± 0.9 min-1, P < 0.05) and increased the Michaelis-Menten constant KM (204 ± 6 nM 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.

Computer simulation of transfusion with different blood product ratios in modern massive transfusion protocols.

Modern massive transfusion protocols call for early plasma and platelets to patients presenting with hemorrhagic shock. The packed red blood cell (PRBC):plasma:platelet ratio generally ranges from 1:1:1 to 3:1:1, but the ideal ratio remains controversial. We aimed to determine the effects of different resuscitation strategies and blood product ratios on hematocrit, platelet and fibrinogen concentrations (FC) during resuscitation. Assuming: pre-insult blood volume 5 L; hematocrit 0.4, FC = 100%, platelet count 400 × 109/L; predetermined constant values for each blood product unit (volume, hematocrit, FC, platelet number); and transfusion rate to maintain euvolemia, we simulated different resuscitation strategies using a computer-based hemorrhage model. When crystalloids are administered to restore an acute 30% blood loss, the initial hematocrit, platelets and FC are adequate, and remain physiologic when further resuscitation is carried out with 1:1:1. Higher transfusion ratios increase the hematocrit at the expense of proportional drops in FC and platelets. When crystalloids and PRBCs (1500 mL) are administered to restore an acute 60% blood loss, the FC drops to 39%. Further resuscitation with 1:1:1 (but not with 2:1:1 or 3:1:1) increases the FC while maintaining the hematocrit and platelets within physiologic range. When blood products (1-3:1:1) are administered to restore an acute 60% blood loss, the initial hematocrit, platelets and FC are at adequate levels, but remain within physiologic range only when 1:1:1 (but not 2:1:1 or 3:1:1) is implemented for further resuscitation. Notably, platelet concentration consistently drops in all simulated scenarios reaching dangerously low levels particularly with high blood loss/transfusion rates and with higher transfusion ratios. The FC does not always drop by the same proportion with higher ratios probably because it is based on plasma concentration and is thus "cushioned" by the reduction in plasma volume as the hematocrit rises with higher transfusion ratios. In summary, computer simulation suggests that in non-severe shock hemorrhage, the differences between 1-3:1:1 transfusion ratios during initial resuscitation may be small. In severe shock, however, 1:1:1 results in the most physiologic hematocrit, FC and platelet concentration and is, therefore, desirable.

Behçet's syndrome as a tool to dissect the mechanisms of thrombo-inflammation: clinical and pathogenetic aspects.

Behçet's syndrome (BS) is a complex disease with different organ involvement. The vascular one is the most intriguing, considering the existence of a specific group of patients suffering from recurrent vascular events involving the venous and, more rarely, the arterial vessels. Several clinical clues suggest the inflammatory nature of thrombosis in BS, especially of the venous involvement, thus BS is considered a model of inflammation-induced thrombosis. Unique among other inflammatory conditions, venous involvement (together with the arterial one) is currently treated with immunosuppressants, rather than with anti-coagulants. Although many in-vitro studies have suggested the different roles of the multiple players involved in clot formation, in-vivo models are crucial to study this process in a physiological context. At present, no clear mechanisms describing the pathophysiology of thrombo-inflammation in BS exist. Recently, we focused our attention on BS patients as a human in-vivo model of inflammation-induced thrombosis to investigate a new mechanism of clot formation. Indeed, fibrinogen displays a critical role not only in inflammatory processes, but also in clot formation, both in the fibrin network and in platelet aggregation. Reactive oxygen species (ROS)-derived modifications represent the main post-translational fibrinogen alterations responsible for structural and functional changes. Recent data have revealed that neutrophils (pivotal in the pathogenetic mechanisms leading to BS damage) promote fibrinogen oxidation and thrombus formation in BS. Altogether, these new findings may help understand the pathogenetic bases of inflammation-induced thrombosis and, more importantly, may suggest potential targets for innovative therapeutic approaches.

Habu coagulotoxicity: Clinical implications of the functional diversification of Protobothrops snake venoms upon blood clotting factors.

Venom can affect any part of the body reachable via the bloodstream. Toxins which specifically act upon the coagulation cascade do so either by anticoagulant or procoagulant mechanisms. Here we investigated the coagulotoxic effects of six species within the medically important pit viper genus Protobothrops (Habu) from the Chinese mainland and Japanese islands, a genus known to produce hemorrhagic shock in envenomed patients. Differential coagulotoxicity was revealed: P. jerdonii and P. mangshanensis produced an overall net anticoagulant effect through the pseudo-procoagulant clotting of fibrinogen; P. flavoviridis and P. tokarensis exhibit a strong anticoagulant activity through the destructive cleavage of fibrinogen; and while P. elegans and P. mucrosquamatus both cleaved the A-alpha and B-beta chains of fibrinogen they did not exhibit strong anticoagulant activity. These variations in coagulant properties were congruent with phylogeny, with the closest relatives exhibiting similar venom effects in their action upon fibrinogen. Ancestral state reconstruction indicated that anticoagulation mediated by pseudo-procoagulant cleavage of fibrinogen is the basal state, while anticoagulation produced by destructive cleavage of fibrinogen is the derived state within this genus. This is the first in depth study of its kind highlighting extreme enzymatic variability, functional diversification and clotting diversification within one genus surrounding one target site, governed by variability in co-factor dependency. The documentation that the same net overall function, anticoagulation, is mediated by differential underlying mechanics suggests limited antivenom cross-reactivity, although this must be tested in future work. These results add to the body of knowledge necessary to inform clinical management of the envenomed patient.

Extravascular fibrinogen in the white matter of Alzheimer's disease and normal aged brains: implications for fibrinogen as a biomarker for Alzheimer's disease.

The blood-brain barrier (BBB) regulates cerebrovascular permeability and leakage of blood-derived fibrinogen. Dysfunction of the BBB has been associated with cerebral arteriolosclerosis small vessel disease (SVD) and white matter lesions (WML). Furthermore, BBB dysfunction is associated with the pathogenesis of Alzheimer's disease (AD) with the presence of CSF plasma proteins suggested to be a potential biomarker of AD. We aimed to determine if extravascular fibrinogen in the white matter was associated with the development of AD hallmark pathologies, i.e., hyperphosphorylated tau (HPτ) and amyloid-ß (Aß), as well as SVD, cerebral amyloid angiopathy (CAA) and measures of white matter damage. Using human post-mortem brains, parietal tissue from 20 AD and 22 non-demented controls was quantitatively assessed for HPτ, Aß, white matter damage severity, axonal density, demyelination and the burden of extravascular fibrinogen in both WML and normal appearing white matter (NAWM). SVD severity was determined by calculating sclerotic indices. WML- and NAWM fibrinogen burden was not significantly different between AD and controls nor was it associated with the burden of HPτ or Aß pathology, or any measures of white matter damage. Increasing severity of SVD was associated with and a predictor of both higher WML- and NAWM fibrinogen burden (all P < 0.05) in controls only. In cases with minimal SVD NAWM fibrinogen burden was significantly higher in the AD cases (P < 0.05). BBB dysfunction was present in both non-demented and AD brains and was not associated with the burden of AD-associated cortical pathologies. BBB dysfunction was strongly associated with SVD but only in the non-demented controls. In cases with minimal SVD, BBB dysfunction was significantly worse in AD cases possibly indicating the influence of CAA. In conclusion, extravascular fibrinogen is not associated with AD hallmark pathologies but indicates SVD, suggesting that the presence of fibrinogen in the CSF is not a surrogate marker for AD pathology.