Reduced Fibrinogen Levels Research Articles

The plasminogen receptor, Plg-RKT, plays a role in inflammation and fibrinolysis during cutaneous wound healing in mice

Wound healing is a complex physiologic process that proceeds in overlapping, sequential steps. Plasminogen promotes fibrinolysis and potentiates the inflammatory response during wound healing. We have tested the hypothesis that the novel plasminogen receptor, Plg-RKT, regulates key steps in wound healing. Standardized burn wounds were induced in mice and time dependence of wound closure was quantified. Healing in Plg-RKT−/− mice was significantly delayed during the proliferation phase. Expression of inflammatory cytokines was dysregulated in Plg-RKT−/− wound tissue. Consistent with dysregulated cytokine expression, a significant delay in wound healing during the proliferation phase was observed in mice in which Plg-RKT was specifically deleted in myeloid cells. Following wound closure, the epidermal thickness was less in Plg-RKT−/− wound tissue. Paradoxically, deletion of Plg-RKT, specifically in keratinocytes, significantly accelerated the rate of healing during the proliferation phase. Mechanistically, only two genes were upregulated in Plg-RKT−/− compared with Plg-RKT+/+ wound tissue, filaggrin, and caspase 14. Both filaggrin and caspase 14 promote epidermal differentiation and decrease proliferation, consistent with more rapid wound closure and decreased epidermal thickness during the remodeling phase. Fibrin clearance was significantly impaired in Plg-RKT−/− wound tissue. Genetic reduction of fibrinogen levels to 50% completely abrogated the effect of Plg-RKT deletion on the healing of burn wounds. Remarkably, the effects of Plg-RKT deletion on cytokine expression were modulated by reducing fibrinogen levels. In summary, Plg-RKT is a new regulator participating in different phases of cutaneous burn wound healing, which coordinately plays a role in the interrelated responses of inflammation, keratinocyte migration, and fibrinolysis.

PULMONARY EMBOLISM AND RIGHT HEART STRAIN PATTERN IN COVID-19: AN INCIDENTAL FINDING OR A PERSISTENT PATTERN?

SESSION TITLE: Medical Student/Resident Pulmonary Vascular Disease Posters SESSION TYPE: Med Student/Res Case Rep Postr PRESENTED ON: October 18-21, 2020 INTRODUCTION: Coronavirus disease 2019 (CVOD-19) is presumed to be a prothrombotic state however, there is no concrete evidence supporting therapeutic anticoagulation in this disease. Here, we present a case of a 67-year old male diagnosed with COVID-19 who developed an acute pulmonary embolism and right ventricular strain pattern during hospitalization and was treated with therapeutic Lovenox. CASE PRESENTATION: A 67-year-old male who had tested positive for COVID-19 one week ago presented to the hospital with shortness of breath, fever (T 102.8), and generalized body aches. His oxygen saturation worsened, and he was put on the nasal cannula, later transitioning to non-rebreather, and finally intubation for not maintaining saturation. Computerized tomography (CT) scan of the chest on admission revealed bilateral, bibasilar and sub-pleural ground-glass opacities. (Figure 1) One week later, he became hypotensive, tachycardiac, and hypoxic on the same ventilator settings. Bedside echocardiogram revealed right ventricular dilatation and akinesis of the mid free wall. (Figure 2) A provisional diagnosis of pulmonary embolism was made and he was treated with therapeutic Lovenox. CT scan of the chest with contrast confirmed the diagnosis of pulmonary embolism. (Figure 3) His hospital course was complicated by pneumonia treated which was treated with antibiotics. He was discharged to a nursing home with a tracheostomy tube and Eliquis on the third week of hospitalization. DISCUSSION: Emerging as a cluster of pneumonia cases in Wuhan, China in December 2019, COVID-19 has spread across the world. It attaches to angiotensin-converting enzyme 2 expressed in the lungs, heart, and gastrointestinal tract. [1] Widespread microvascular thrombi in the pulmonary circulation as evidenced by autopsy studies cause profound hypoxia by a ventilation-perfusion mismatch in the lungs. The proposed mechanism of COVID-19 thrombosis includes hypercoagulable state and cytokine-mediated damage. [2] It is believed that 50 % of these patients have elevated D-dimer levels that in combination with elevated prothrombin time and reduction in fibrinogen level have been associated with increased mortality.[3] Some enterprises are therapeutically anticoagulating these patients based on these coagulation parameters while others are not. CONCLUSIONS: COVID-19 increases the incidence of microvascular and microvascular thrombotic complications. Worsening respiratory status not explained by radiological changes in the lung fields, and especially in conjunction with high titers of D-dimers, should raise the suspicion for pulmonary embolism. More studies are needed to evaluate the need for therapeutic anticoagulation in these patients without objective evidence of thrombosis. Reference #1: Yang XL, Wang XG, Hu B, Zhang L, Zhang W, et al. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature. 2020. https://doi.org/10.1038/s41586-020-2012-7. Reference #2: Mei H., Hu Y. (2020) Characteristics, causes, diagnosis and treatment of coagulation dysfunction in patients with COVID-19. Zhonghua Xue Ye Xue Za Zhi 41:E002. Reference #3: ang N, Li D, Wang X, Sun Z. Abnormal coagulation parameters are associated with poor prognosis in patients with novel coronavirus pneumonia. Journal of Thrombosis and Haemostasis. 2020 Apr 1. DISCLOSURES: No relevant relationships by John Madara, source=Web Response No relevant relationships by Sohaib Roomi, source=Web Response

The clinical efficacy of AG NPP709 (Synatura®) in patients with chronic bronchitis type stable chronic obstructive pulmonary disease.

BackgroundAG NPP709 (Synatura®) has been demonstrated to be efficacious in decreasing cough and sputum in patients with acute upper respiratory infection and chronic inflammatory bronchitis. The aim of this study was to evaluate the efficacy of AG NPP709 in patients with chronic bronchitis type chronic obstructive pulmonary disease (COPD).MethodsThis was a prospective, open-label, single-arm clinical trial (NCT03623282). Chronic bronchitis type COPD patients aged >40 years were enrolled. The primary endpoint was improvement on the CAT scores between the baseline visit and week 12. The secondary endpoints were the effect of AG NPP709 on the pulmonary function and systemic inflammation, as indexed by CRP, fibrinogen, IL-6, TNF-α and IL-33 levels.ResultsThirty patients were enrolled. All patients were male, and their mean age was 71.93±7.93 years. The mean post-bronchodilator forced expiratory volume in 1 second/forced vital capacity (FEV1/FVC) ratio was 58.63±11.40, and FEV1 (%) was 75.93±20.42. The mean total CAT score was 14.77±7.14. Of these patients, 26 were followed up after 3 months. A significant improvement was observed in the total CAT score (from 14.38±6.62 to 12.73±6.60, P=0.005). Fibrinogen level decreased significantly (P=0.013). No serious adverse events occurred.ConclusionsAG NPP709 improved the quality of life, as represented by the CAT score, in patients with chronic bronchitis type COPD, and significantly reduced fibrinogen levels. These results suggest that AG NPP709 is efficacious and safe in patients with chronic bronchitis type COPD.

The Plasminogen Receptor, Plg‐RKT, Regulates Inflammation and Fibrinolysis During Wound Healing

Plasminogen is a key regulatory protein that functions to both promote fibrinolysis and to potentiate the inflammatory response during wound healing. Here we have tested the hypothesis that the novel plasminogen receptor, Plg‐RKT, regulates distinct steps in the wound healing process.MethodFull‐thickness standardized burn wounds (1 cm in diameter) were induced in mice and the wound area was quantified at different time points. Wound tissue was subjected to qPCR and ELISA for cytokine expression and immunohistochemistry and Western blotting for fibrin deposition and leukocyte recruitment.ResultsFrom day 9 after injury, healing in Plg‐RKT−/− mice was significantly delayed compared with healing in Plg‐RKT+/+ littermates. Expression of cytokines, IL‐1β, CCL‐20, TNFα, and IL‐10 was dysregulated in Plg‐RKT−/− tissue although no genotype‐dependent differences in recruitment of macrophages or neutrophils to the wound area were detected. Consistent with dysregulated cytokine expression, a significant delay in wound healing during the proliferation phase (days 9 and 10) was observed in wound tissue from mPlg‐RKT−/− mice in which Plg‐RKT was specifically deleted in myeloid cells. Regarding fibrinolysis, fibrin clearance was significantly impaired in Plg‐RKT−/− wound tissue. Genetic reduction of fibrinogen levels of Plg‐RKT−/− mice to 50% completely abrogated the effect of Plg‐RKT deletion on healing of burn wounds and, remarkably, effects of Plg‐RKT deletion on cytokine expression were modulated by the reduction in fibrinogen levels. In conclusion, Plg‐RKT plays a key role in wound healing by regulating both fibrinolysis and the inflammatory response and fibrin plays a role in regulating Plg‐RKT function on myeloid cells.Support or Funding InformationUnited States National Institutes of Health grants HL 081046 to LAM and HL 149511 to LAM and RJP and Merit Review Award #5I01BX002026 from the U.S. Department of Veterans Affairs to RJP.

Effect of Tranexamic Acid on Coagulation and Fibrin Clot Properties in Children Undergoing Craniofacial Surgery.

Craniosynostosis surgery in small children is very often associated with a high blood loss. Tranexamic acid (TXA) reduces blood loss during this procedure, although the potential underlying coagulopathy in these children is not known in detail. Objective was to determine the nature of any coagulopathy found during and after craniosynostosis surgery and to characterize the effect of TXA on fibrin clot formation, clot strength, and fibrinolysis. Thirty children received either TXA (bolus dose of 10 mg/kg followed by 8 hours continuous infusion of 3 mg/kg/h) or placebo. Dynamic whole blood clot formation assessed by thromboelastometry, platelet count, dynamic thrombin generation/thrombin-antithrombin, clot lysis assay, and fibrinogen/factor XIII (FXIII) levels were measured. Additionally, clot structure was investigated by real-time live confocal microscopy and topical data analysis. Increased ability of thrombin generation was observed together with a tendency toward shortened activated partial thromboplastin time and clotting time. Postoperative maximum clot firmness was higher among children receiving TXA. FXIII decreased significantly during surgery in both groups.Resistance toward tissue plasminogen activator-induced fibrinolysis was higher in children that received TXA, as evidenced by topical data analysis and by a significant longer lysis time. Fibrinogen levels were higher in the TXA group at 24 hours. A significant coagulopathy mainly characterized by changes in clot stability and not parameters of thrombin generation was reported. Tranexamic acid improved clot strength and reduced fibrinolysis, thereby avoiding reduction in fibrinogen levels.

Thrombin generation test with the calibrated automated thrombogram and anticoagulant activity of Mentha crispa.

: Evaluate the in-vitro effect of Mentha crispa extract on blood coagulation, compare the conventional coagulometric tests with thrombin generation test (TGT), and study the qualitative micromolecular composition of M. crispa. Extract of M. crispa was incubated with plasma and used in the coagulometric tests: prothrombin and activated partial thromboplastin times, fibrinogen, and TGT. A phytochemical prospection was performed to evaluate the chemical composition of this extract. The extract was efficient in prolonging prothrombin time and activated partial thromboplastin time, and reducing fibrinogen levels and TGT parameters, indicating that the extract of M. crispa inhibited the intrinsic and extrinsic pathways of blood coagulation. The results obtained in TGT are in agreement with the results of conventional coagulometric tests and the in-vitro anticoagulant activity of M. crispa suggests that its use by patients using oral anticoagulants deserves caution.

Association of fibrinogen and D‑dimer levels with severity of acute coronary syndromes

Background: Acute coronary syndromes (ACSs) are the primary cause of mortality worldwide. The aim of the study was to assess the as‑sociations of serum fibrinogen and plasma D‑dimer levels with angiographic severity of atherosclerotic lesions as well as the presence of in‑hospital complications and complications at 30‑day follow‑up in patients with ACS. Methods: This was a prospective study including 107 patients with ACS. Severity of CAD was assessed by the Gensini score. Correlations of D‑dimer and fibrinogen levels with complica‑tions such as heart failure, arrhythmia, recurrent angina, and cardiac death were assessed using the Pearson correlation coefficient and the receiver operating characteristic curve analysis. Results: The mean age of patients was 61±10.9 years. Mean serum fibrinogen levels were higher in individuals with severe left ventricular (LV) dysfunction than in those with moderate and mild LV dysfunction (444 mg/dl, 404 mg/dl, and 330 mg/dl, respectively). Similarly, the mean plasma D‑dimer level was higher in individuals with severe ACS (1.03 μg/ml) than in those with moderate (1.88 μg/ml) and mild ACS (3.5 μg/ml). Conclusion: Our study revealed that patients with higher serum fibrinogen levels tend to have more severe ACS, greater LV dysfunction, and a higher rate of complications. Therapies aimed at reducing fibrinogen levels might help reduce mortality and morbidity in patients with ACS.

Autoimmune Thrombotic Thrombocytopenic Purpura, Disseminated Intravascular Coagulation, Haemophagocytic Lympho Histiocytosis—(Following Corona Virus Infection), Secondary to Fragmented Germ Cells {Liberal Global Abortions, Contraception—(Red Riverways, Blood Moon, Weakened Earth’s Magnet)} and Absence of Abstinence—Retrospective Analysis: Proof of Concept Study

Background: Autoimmunity has increased significantly, alarmingly secondary to global contraception, abortion, fragmenting germ cells; herewith three types of life threatening manifestations, are analysed. Case-1: 28-year-old afebrile lady, vegetarian, presented to emergency with impaired responsiveness, deep jaundice, menorrhagia of one day duration and she had been taking native treatment with leaves for primary infertility for the past five years; she had a few ecchymotic patches, investigations showed severe anaemia, thrombocytopenia, haemolytic jaundice, peripheral smear confirmed thrombotic thrombocytopenic purpura; she developed profound hematuria; she received treatment with intravenous immunoglobulin, fresh frozen plasma, platelet transfusions, packed red blood cells transfusions, plasmapheresis was planned; she improved gradually. In her sincere effort to conceive a baby, for the past five years, due to cultural ignorance which teaches only three days abstinence, instead of seven days abstinence after last menstrual periods, exposure of germ cells to raw endometrial surface, from 4th to 7th days of menstrual cycle, resulted in the cumulative autoimmunity-thrombotic thrombocytopenic purpura status. Case-2: 24-year-old lady who had undergone puerperal sterilisation, on the fourth day after last menstrual periods, presented, with unresponsiveness, to emergency, her investigations revealed prolonged prothrombin time, prolonged activated partial thromboplastin time, more than a minute, thrombocytopenia, reduced fibrinogen levels suggesting disseminated intravascular coagulation; her ultra sonogram abdomen showed, absence of gestational sac in the uterus; she received fresh frozen plasma transfusions, but succumbed within few hours. With puerperal sterilisation, [non evidence based medical practice], 20 million germ cells are fragmented to chromatid breaks, a-centric fragments, ring chromosomes, which get exposed to partly regenerated endometrial surface, on the fourth day after last menstrual periods, due to lack of awareness of abstinence upto seven days, initiating severe cumulative autoimmunity, resulting in fatal disseminated intravascular coagulation. Case-3: Fatal haemo-phagocytic lympho-histiocytosis-soaring autoimmunity, detected in the victim host of corona virus infection, correlated directly with extensive tissue damage produced in the victim host, by prior surgical abortions. Conclusion: Cases one and two followed absence of abstinence, with exposure of germ cells, to partly healed endometrial surface in the former culminating in thrombotic thrombocytopenic purpura; in the latter by exposure of fragmented 20 million germ cells [by contraception] to partly healed endometrial surface, culminating in fatal disseminated intravascular coagulation. In case three fatal autoimmune hemophagocytic lymphohistiocytosis followed corona virus infection, secondary to extensive tissue damage, of liberal global abortions. Red Russian riverways, is another evidence for global warming due to global aborted blood pollution, melting Perma-Frost crashing the Diesel tanks, with oil spill reflecting on aborted blood polluted river, turns bright red, similar to blood moon by abortion polluted air. Innumerable human lives with their iron of blood flowing within blood vessels, repelling magnet of earth, is mandatory to strengthen earth’s magnet. Arctic ozone hole closed this May, environmental pollution cleared, by absence of global abortions, contraception, and their ozone depleting blood pollution, due to lock down of corona virus pandemic.

PEG-L-CHOP treatment is safe and effective in adult extranodal NK/T-cell lymphoma with a low rate of clinical hypersensitivity

BackgroundThe combination of chemotherapy and L-asparaginase (L-ASP) treatment significantly increased survival rate in an adult patient with extranodal natural killer (NK)/T-cell lymphoma (NKTCL). However, hypersensitivity reactions of L-ASP in some patients limited its application. Polyethylene glycol-conjugated asparaginase (PEG-ASP) has a lower immunogenicity and longer circulating half-life than unconjugated L-ASP, and has been reported to be effective and well-tolerated in children with acute lymphoblastic leukemia. Cyclophosphamide, hydroxydaunorubicin (doxorubicin), oncovin (vincristine), and prednisolone (CHOP) is the most common chemotherapy for non-Hodgkin lymphoma. In this report, we sought to study the efficacy and safety of PEG-L- CHOP in NKTCL in adult Chinese patients.MethodsOur study is a prospective, multi-center, open-label clinical trial. Patients with newly diagnosed adult NKTCL and an ECOG performance status of 0 to 2 were eligible for enrollment. Treatment included six cycles of PEG-L-CHOP regimen. Radiotherapy was scheduled after 2–4 cycles of PEG-L-CHOP regimen, depending on the stage and primary anatomic site.ResultsWe enrolled a total of 33 eligible patients. All 33 patients completed 170 cycles of chemotherapy combined with radical radiotherapy. The overall response rate was 96.9% (32/33) with 75.8% (25/33) achieving complete responses and 21.2% (7/33) achieving partial responses. The overall survival (OS) at 1, 2, 3-year were 100, 90.61 and 80.54%, respectively. The major adverse effects were bone marrow suppression, reduction of fibrinogen level, liver dysfunction, and digestive tract toxicities. No allergic reaction and no treatment-related mortality or severe complications were recorded.ConclusionsPEG-L-CHOP chemotherapy in combination radiotherapy is safe and durably effective treatment for adult extranodal NK/T-cell lymphoma with fewer allergic reactions.This study was approved by the Peking University Beijing Cancer Hospital Ethics Review Committee (reference number: 2011101104). The clinical trial registration number ChiCTR1800016940 was registered on July 07, 2018 at the Chinese Clinical Trial Registry (http://www.chictr.org.cn/index.aspx). The clinical trial was registered retrospectively.

Coagulopathic side effect of L-asparaginase on fibrinogen level in childhood acute lymphoblastic leukemia during induction phase

BackgroundL-asparaginase is chemotherapeutic agent commonly used in treatment of Acute Lymphoblastic Leukemia. Thromboembolic and to a lesser extent bleeding events are serious complications associated with administration of this drug during the induction phase in ALL especially in children resulting from reduced synthesis of proteins such as Antithrombin & fibrinogen. ObjectiveEvaluation of the coagulopathic side effect of L-asparaginase during the induction phase in treatment of ALL in children by measuring fibrinogen levels before & after administration of this drug. Subjects & methodAn interventional prospective study was performed on 30 newly diagnosed children with ALL, all of them received 9 cycles of L-asparaginase during the induction phase according to UKALL 2003 Protocol.Fibrinogen was measured before & after treatment with L-asparaginase and the results are compared to a control group of 30 healthy children age & sex matched. ResultsReduced fibrinogen level is observed in 13 out of 30 patients (43.3%) after treatment with L-asparaginase ranging from 25 to 110 mg/dl with means fibrinogen before & after treatment of 252 ± 16.40 mg/dl & 158.97 ± 17.88 mg/dl respectively (p < .0001). ConclusionSignificant reduction in fibrinogen level occurred in childhood ALL patients after treatment with L-asparaginase.

Tocilizumab treatment in patients with rheumatoid arthritis is associated with reduced fibrinogen levels and increased blood loss after total knee arthroplasty

Objective: Since IL-6 has been associated with activation of the coagulation cascade and upregulation of fibrinogen transcription, we retrospectively tested the hypothesis that patients with rheumatoid arthritis (RA) treated with tocilizumab (TCZ) may lose more blood when undergoing total knee arthroplasty (TKA).Methods: This study included 115 RA patients who underwent primary TKA and were preoperatively tested for fibrinogen levels. The blood volume of each patient was calculated using the Nadler formula, and estimated blood loss after TKA was calculated as the change between pre-operative and post-operative hematocrits. If salvaged blood was reinfused, the volume was measured and added to the volume of the estimated blood loss.Results: We observed that patients treated with TCZ had significantly lower pre-operative fibrinogen levels than those not treated with TCZ (190.0 mg/dL versus 347.0 mg/dL, respectively; p = .00018). We also observed a statistically significant increase in mean total volume of estimated blood loss after TKA in RA patients who had been treated with TCZ compared with those not treated with TCZ (797.1 mL versus 511.4 mL, respectively; p = .0039).Conclusion: TCZ treatment in patients with RA may increase the risk of blood loss after TKA because of decreased fibrinogen levels.

Physical Activity Alters Inflammation in Older Adults by Different Intensity Levels.

To examine the influence of reallocating time spent at different objectively measured physical activity (PA) behaviors on markers of systemic inflammation in older women with different levels of metabolic risk. Accelerometer-based monitoring of PA was conducted in a population of community-dwelling older women (n = 111; age, 65-70 yr) for determination of daily sedentary time, time in light PA (LPA) and moderate-to-vigorous PA (MVPA). Blood samples were collected for the assessment of the systemic inflammatory markers C-reactive protein (CRP), fibrinogen, and adiponectin. Metabolic risk was assessed by standardized procedures based on definitions for the metabolic syndrome. Data were analyzed by linear regression models based on isotemporal substitution analysis. Reallocating 30 min of sedentary time with either time in LPA (β = -0.47; P < 0.05) or MVPA (β = -0.42; P < 0.05) was related to reduced fibrinogen level, whereas no corresponding effect was evident when shifting time in LPA with time in MVPA, while holding sedentary time constant. In contrast, reallocating a 30-min period in sedentary (β = -0.70; P < 0.01) or LPA (β = -0.71; P < 0.01) with MVPA was associated with a significant reduction in CRP level, whereas no impact on CRP was observed when a period of sedentary behavior was replaced with LPA. Importantly, all significant influences on fibrinogen and CRP by displacement of different PA behaviors remained after adjustment for metabolic risk status among participants. No significant associations with adiponectin were observed. Altogether, this work supports the existence of different intensity thresholds mediating beneficial effects of PA on important clinical markers of systemic inflammation in older women across different stages of disease prevention.

Successful treatment with dabigatran for consumptive coagulopathy associated with extensive vascular malformations.

Vascular malformation is occasionally complicated by consumptive coagulopathy, known as localized intravascular coagulopathy (LIC), which is characterized by a reduced fibrinogen level, an elevated D-dimer level and a normal platelet count. We report the case of a 17-year-old Japanese girl who presented with LIC secondary to extensive vascular malformations, whose condition had progressed to disseminated intravascular coagulation (DIC). She suddenly presented with severe anaemia, despite the absence of obvious bleeding, and she began to require regular red blood cell (RBC) transfusions. As she was suffering from paroxysmal atrial fibrillation, we treated her with dabigatran, after obtaining informed consent. Immediately after the administration of dabigatran, the results of clotting tests improved dramatically. Seven months later, she has not required any RBC transfusions, and the dabigatran treatment has been well tolerated. The present case report suggests that dabigatran may be a useful treatment option for patients with DIC associated with vascular malformations.

Fibrinolysis: A Critical Determinant in Musculoskeletal Tissue Repair

For over a century, the formation of a fibrin clot has been considered to be an initial and fundamental phase of musculoskeletal tissue repair. Following a severe injury, accumulation of fibrin clots is necessary to stop bleeding, yet recent work in our lab has demonstrated that proper healing of both bone and skeletal muscle is dependent on the temporal nature of fibrin and the fibrinolytic mechanisms rather than fibrins presence in damaged tissues.Fibrinolysis in Fracture HealingFibrin, the principle polymer protein of the coagulation system, was long believed essential to fracture healing by providing the initial scaffold for osteo‐regenerative mediators. Hence, many principles of musculoskeletal injury care and pharmaceuticals have been developed to provide a fibrin matrix in the fracture bed thought to represent the initial scaffold of fracture healing. However, we have recently demonstrated that fibrin is not essential for fracture healing, rather, persistent fibrin, which was not effectively removed via fibrinolytic mechanism, impaired fracture healing and inhibited union. Specifically, by creating a mid‐shaft femur fracture on 8 week old male mice (Wild type (WT)); n= 15, plasminogen deficient (Plg−/−) which cannot remove fibrin; n= 14, and fibrinogen deficient (Fbg−/−) which cannot make a fibrin clot; n=6) we revealed that WT and Fbg (−/−) mice formed ossified bridging callus by 3 weeks post fracture which was almost completely remodeled and all united by 6 weeks post fracture. Alternatively, Plg (−/−) mice failed to develop an organized mineralized callus and failed to unite or remodel the disorganized matrix (Figure 1A). Consistent with this finding, there was abundant avascular cartilage in Plg (−/−) mice at 2 weeks post fracture as compared to WT mice. Fibrin and CD31 immunohistochemical (IHC) staining showed abundant fibrin interposed between the avascular cartilage and vascularized bone (Figure 1B and C). Finally, reducing fibrinogen levels in Plg (−/−) mice with a targeted antisense oligonucleotide partially rescued the fracture healing in these mice. Considering that many conditions which result in pathologic fracture healing such as diabetes, smoking and aging all have impaired fibrinolysis resulting in fibrin accumulation in tissues, these results may provide valuable insight into novel means of improving fracture healing in these populations by targeting fibrin degradation.Fibrinolysis in Skeletal Muscle HealingLike bone, a fibrin meshwork forms in damaged skeletal muscle following injury, yet is resolved by the main protease of the fibrinolytic system, plasmin, to allow for timely and robust tissue regeneration. Through recent work in our lab, we have demonstrated that the protease plasmin, in addition to its canonical fibrinolytic function, also works to prevent the aberrant formation of mineralization within soft tissues. Specifically, extensive or persistent calcium phosphate deposition within soft tissues after a traumatic injury or major orthopedic surgery can result in pain and loss of joint function. The pathophysiology of soft tissue calcification, including dystrophic calcification and heterotopic ossification (HO), is poorly understood. Yet, we have demonstrated that after muscle injury, insufficient plasmin activity can lead to the formation of dystrophic calcification (Figure 2A). Additionally, we have found that if the dystrophic calcification is persistent within damaged tissues, it can become organized into mature bone, known as HO. Importantly, we determined that downregulating the primary inhibitor of plasmin (α2‐antiplasmin) pharmacologically prevented dystrophic calcification and subsequent HO in vivo (Figure 2A and Figure 2B). Since plasmin also supports bone homeostasis and fracture repair, increasing plasmin activity represents the first pharmacologic strategy to prevent soft tissue calcification without adversely affecting systemic bone physiology or concurrent muscle and bone regeneration.Together these investigations have shifted the paradigm of the role of fibrinolysis in musculoskeletal tissue repair. By utilizing animal models that effectively phenocopy different forms of human musculoskeletal injury, we aspire to translate these findings to the development of novel therapeutics aimed at improving tissue regeneration following a severe injury.Support or Funding InformationFunding for this work was provided by the Vanderbilt University Medical Center Department of Orthopaedics and Rehabilitation, The Caitlin Lovejoy Foundation, RO3 (1RO3AR065762), Orthopedic Pilot Program Award, The Musculoskeletal Transplant Fund, HHMI Student Fellowship, The Orthopaedic Trauma Association, Vascular Biology Training Grant‐Postdoctoral Funding, and the Predoctoral Pharmacology Training Grant (T32 GM007628).

Reducing Fibrinogen through Platelet Additive Solutions Improves Mitochondrial Function and Reduces Reactive Oxygen Species in Stored Platelets

BackgroundThe platelet storage lesion is mitigated by refrigeration in platelet additive solutions (PAS), but questions remain regarding effects of cold storage (4°C) on mitochondrial function. Understanding underlying mechanisms responsible for enhanced hemostatic function of 4°C-stored PAS platelets will be critical for such improvements in platelet transfusion. We hypothesized that storage of platelet concentrates at 4°C leads to GPIIb-IIIa activation and thus aggregate formation through fibrinogen binding, and that this could be prevented by storing platelets in platelet additive solution (PAS) without compromising platelet function.MethodsApheresis platelets in plasma (AP) or apheresis platelets in PAS were stored at 22°C or 4°C for up to 15 days. Measurements include platelet counts, blood gases, flow cytometry analysis, intracellular reactive oxygen species (ROS) levels, and high resolution respirometry. To explore the effect of fibrinogen on storage, platelets were treated with abciximab to inhibit GPIIb-IIIa binding and aggregation. Donor fibrinogen levels were measured before being collected in PAS; samples were separated into minibags for comparison of control 4°C PAS-stored platelets to 4°C PAS-stored platelets with fibrinogen added back to original donor levels (PAS-FBN).ResultsMitochondrial respiration was lower in platelets stored at 4°C compared to 22°C in both AP and PAS; however, PAS-stored platelets retained lower mitochondrial respiration over the time course. Additionally, maximal mitochondrial oxygen utilization (oxidative burst capacity) was better preserved in 4°C-stored platelets; PAS demonstrated less overall mitochondrial exhaustion at 4°C compared to 4°C-AP samples (Figure 1). PAS-FBN resulted in reduced oxidative burst capacity over 15 days of storage which was consistent with loss of platelet function observed through flow cytometry analysis including increased phosphatidylserine expression, microparticles, and GPIb shedding. Mitochondrial intracellular ROS levels were lower in all 4°C-stored samples compared to their respective RT-stored groups. PAS-FBN resulted in significantly lower platelet counts (Day 5: BL= 1.1x106±.04plt/µL, PAS = 1.1 x106±.06plt/µL, PAS-FBN = .61 x106±.06plt/µL; P<0.001) compared to 4°C PAS-stored platelets and displayed marked increases in visible aggregates in the storage bag. PAS-stored platelets resulted in significantly lower levels of intracellular ROS compared to AP at both RT and 4°C conditions and PAS-FBN (P<0.05). 4°C-AP-stored samples treated with abciximab (AP-Ab) rescued the loss in platelet count commonly encountered in 4°C-AP storage (Day 5: BL = 1112±18, AP-4°C = 720±77, AP-Ab = 1085±28;P<0.001) and resulted in less intracellular ROS compared to untreated 4°C-AP samples at Day 5 (Figure 2).DiscussionMitochondrial damage and ROS production may contribute to loss of platelet viability during storage, whereas cold storage is known to preserve platelet function. Our previous work has shown that use of PAS in stored platelets prevents aggregate formation, enhances platelet function, results in less oxidant stress, and preserves both mitochondrial function and potential compared to AP-stored platelets. Here we demonstrate that reduction in fibrinogen levels of 4°C PAS -stored platelets plays a key role in the underlying mechanism for PAS-stored platelet enhancement. Taken together, these data indicate that cold storage combined with PAS preserves platelet viability both by maintaining energy potential and preventing degradation due to enhanced ROS. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

An in vitro study of the effects of t-PA and tranexamic acid on whole blood coagulation and fibrinolysis

AimsAcute traumatic coagulopathy is characterised by fibrinolysis and low fibrinogen. It is unclear how much fibrinogenolysis contributes to reduce fibrinogen levels. The study aim was to: investigate in vitro the...

Analysis and Treatment of Multiple Severe Venous Vascular Malformation Syndrome Combined with Coagulopathy.

Analysis and Treatment of Multiple Severe Venous Vascular Malformation Syndrome Combined with Coagulopathy.

Survey of Poly Fibrinogen Levels in Patients With Major Blunt Trauma

Background: Coagulopathy is common after severe trauma or massive bleeding, and many studies have shown the benefits of coadministration of blood and plasma. Efforts are being made to improve the treatment of patients injured by blunt trauma using plasma infusion. Objectives: This study focused on blood fibrinogen levels in patients with severe trauma and bleeding. Patients and Methods: Twenty-eight patients who were admitted to the Rajaee (Emtiaz) Trauma Hospital were studied in this crosssectional survey, which was conducted in August and September of 2013. The patients were male and over 16 years old, with an injury severity score greater than or equal to 16, diastolic blood pressure under 90 mmHg, a minimum 1500 cc of estimated blood loss, and a minimum of 2000 cc crystalloid fluid intake, and patients were not receiving blood or blood products at the time of the traumatic event. Upon patient arrival, blood samples were obtained to measure fibrinogen concentration before starting resuscitation for the patient. Administration of blood and blood products was then performed according to hospital protocol. Results: Over three months, twenty eight patients were admitted with the above criteria. Among these patients, the fibrinogen levels of eight cases (28.57%) were reported as non-measurable. Fibrinogen levels in 10% of the patients were below the acceptable standard of admission to the center, which means that blood fibrinogen levels were lower than normal in a significant number of patients. Conclusions: Severe tissue damage and bleeding significantly reduce fibrinogen levels, and the positive effects of administering plasma to severe trauma patients may be due to the fibrinogen that plasma provides. It is therefore important to discover safer ways to administer fibrinogen. We recommend that the amount of administered fibrinogen be controlled, and that a thromboelastography be conducted for all patients on arrival.

Assessment of Hemostasis after Plasma Exchange Using Rotational Thrombelastometry (ROTEM).

BackgroundTherapeutic plasma exchange (TPE)-based protocols immediately before cadaveric donor kidney transplantation have been extensively used in highly sensitized recipients. Plasma is generally preferred over human albumin as replacement fluid to avoid depletion of coagulation factors and perioperative bleeding. The aim of this study was to estimate bleeding risk after TPE replaced with albumin using rotational thromboelastography (ROTEM).MethodologyTen patients without overt coagulation abnormalities underwent TPE. Standard laboratory coagulation tests (thromboplastin time, activated partial thromboplastin time (aPTT), international normalized ratio (INR), thrombin clotting time, fibrinogen levels and antithrombin activity) were compared with thrombelastometry analysis (EXTEM and INTEM tests) before and after TPE.Principal FindingsTPE significantly reduced fibrinogen levels (482 ± 182 vs. 223 ± 122 mg/dL), antithrombin activity (103 ± 11 vs. 54 ± 11 %), and prolonged aPTT (28 ± 3 vs. 45 ± 8 s), thromboplastin time (108 ± 11 vs. 68 ± 11 %), INR (0.95 ± 0.06 vs. 1.25 ± 0.16), and thrombin clotting time (18 ± 2 vs. 20 ± 3 s). INTEM and EXTEM analyses revealed significantly prolonged clot-formation time and reduced maximum clot firmness.Conclusions/SignificanceTPE replaced with albumin induces significant changes in global hemostasis parameters thus potentially increasing bleeding risk. Therefore, pretransplant TPE should be considered carefully in indicated patients before kidney transplantation. The role of the ROTEM point-of-care test to estimate the risk of bleeding in renal transplantation needs to be evaluated in further studies.

Steroid Therapy Attenuates Acute Phase Reactant Response Among Children on Ventricular Assist Device Support

Hyperfibrinogenemia, which can create a procoagulant milieu, is frequently observed in patients supported with the Berlin EXCOR (Berlin Heart GmbH, Berlin, Germany) ventricular assist device (VAD). We began initiating corticosteroids in patients with systemic inflammatory response syndrome (SIRS) episodes to mitigate hyperfibrinogenemia. We set forth to describe the impact of corticosteroids on the hyperfibrinogenemic state in our institutional experience. Retrospective data was collected on 44 consecutive patients implanted with the Berlin EXCOR VAD from April 15, 2005 through May 6, 2013. Pertinent information was abstracted from the electronic medical record. The reduction of C-reactive protein (CRP) and fibrinogen levels among days from corticosteroid treatment were described. Infections and insulin use were reported based on whether patients received steroids and if steroids were given for SIRS. Over the initial 44 Berlin EXCOR VAD implantations, 14 patients were treated with 21 courses of corticosteroids for SIRS episodes as identified by clinical features and rise in CRP. Treatment with corticosteroids reduced fibrinogen levels by day 2 to a statistically significant degree (p= 0.008). No difference in hyperglycemia or infections occurred among patients receiving corticosteroids for SIRS. Treatment with corticosteroids can potentially mitigate the SIRS response among children supported on the Berlin EXCOR VAD. In patients who received corticosteroids to mitigate inflammation, there was no increase in infections or hyperglycemia requiring insulin administration compared with patients who did not receive steroids.