Cross-linked Fibrin Research Articles

Role of Thromboelastography and Thromboelastometry in Predicting Risk of Hypercoagulability and Thrombosis in Critically Ill COVID-19 Patients: A Qualitative Systematic Review.

Thromboelastography and rotational thromboelastometry are the viscoelastic point of care devices that use whole blood samples to assess coagulation and fibrinolysis. These devices give information from initiation of the coagulation cascade, activation of clotting factors to fibrin cross-linking, and contribution of fibrinogen and platelet to clot strength and clot lysis. Viscoelastic point of care tests are well established in hypocoaguable states like trauma, cardiac surgery, liver transplantation, and their use in critical care settings with coronavirus disease 2019 (COVID-19) is not so well-known. We performed a systematic review of studies on thromboelastography and rotational thromboelastometry and their modifications to assess their role in critically ill patients with COVID-19. Inclusion criteria were any kind of studies using thromboelastography or rotational thromboelastometry during coronavirus disease critical illness published in English. Ninety-three articles, from December 1, 2019, to August 31, 2020, were identified in the initial search, out of which 12 articles (a total of 380 patients) satisfied the inclusion and exclusion criteria. Thromboelastography and rotational thromboelastometry were observed to detect the hypercoagulable changes and fibrinolysis shutdown associated with COVID-19. Hypercoagulability is associated with an increased risk of venous thrombosis and micro-thrombosis. This review identifies the role of thromboelastography and rotational thromboelastometry in studying the mechanisms contributing to coagulopathy and incidence of thrombosis in COVID-19.

Estradiol provokes hypercoagulability and affects fibrin biology: A mechanistic exploration of sex dimorphisms in coagulation.

Sex dimorphisms in coagulation are well established, with female-specific hypercoagulability conferring a survival benefit in the setting of trauma-induced coagulopathy (TIC). The mechanism behind these phenomena remains to be elucidated. We hypothesize that estradiol provokes a hypercoagulable profile and alters clot proteomics and fibrin crosslinking. Whole blood was collected from healthy adult volunteers (n = 30). A battery of thrombelastography (TEG) assays (native, kaolin, platelet-mapping, functional fibrinogen), whole blood thrombin generation, proteomics, and clot structure architecture (via analysis of fibrin crosslinks and fluorescent fibrinogen-visualized clots) were performed after pre-treatment of the blood with physiologic concentrations of beta-estradiol. In addition, a prospective study of coagulation through the menstrual cycle was conducted by collecting blood from women on peak and nadir estrogen days in the standard 28-day menstrual cycle. On TEG, in females, estradiol provoked a hypercoagulable phenotype, specifically a shorter time to clot formation and greater thrombin generation, greater rate of clot propagation and functional fibrinogen, higher clot strength, and diminished clot fibrinolysis. In both males and females, estradiol increased platelet hyperactivity. Similar changes were seen in time to clot formation and clot strength in vivo during peak estrus of the menstrual cycle. On proteomic analysis, in both males and females, estradiol was associated with increases in abundance of several procoagulant and antifibrinolytic proteins. Crosslinking mass spectrometry analysis showed addition of estradiol increased the abundance of several FXIII crosslinks within the FIBA alpha chain in both sexes. Fluorescent fibrinogen analysis revealed a trend toward increased fiber resolvability index after addition of estradiol. Estradiol provokes a hypercoagulable phenotype, affecting time to clot formation, clot propagation, clot strength, clot fibrinolysis, and clot structure. In sum, these data highlight the role of estradiol is driving female-specific hypercoagulability and highlights its potential role as a therapeutic adjunct in resuscitation of TIC.

The effect of the Belmont rapid infuser on cold stored whole blood coagulability

IntroductionWith the large-scale use of whole blood in massive transfusion using rapid infusers/fluid warmers such as the Belmont, questions remain as to whether coagulation potency, platelet number and function are preserved. We aimed to study functional coagulation capacity and cell counts in whole blood before and after infusion through the Belmont rapid infuser utilizing TEG analysis and complete blood counts. MethodsWe evaluated 10 whole blood units before and after infusion through a Belmont Fluid Management System at a set rate of 200 mL/min and a temperature of 37.4 °C. Cell counts and thromboelastography function of the specimens were measured. Parameters were compared utilizing paired Student's t-tests and paired Wilcoxon Rank Sign tests. ResultsPlatelet count, R time, and Maximum amplitude showed significant decreases (defined as p<0.05) after being infused through the Belmont. Hemoglobin, hematocrit, MCV, and alpha angle were not statistically different before and after infusion. ConclusionInfusion of cold stored whole blood in a Belmont infuser, appeared to decrease platelet counts and function as well as activate clotting factors as demonstrated by a shorter R time while not affecting red cell counts or fibrin cross-linking as measured by TEG parameters and cell counts. This suggests that while it is possible to transfuse whole blood through a rapid infuser, platelet quantity and function may be negatively impacted.

Biomarkers of Clot Activation and Degradation and Risk of Future Major Cardiovascular Events in Acute Exacerbation of COPD: A Cohort Sub-Study in a Randomized Trial Population.

Cardiovascular diseases are common in patients with chronic obstructive pulmonary disease (COPD). Clot formation and resolution secondary to systemic inflammation may be a part of the explanation. The aim was to determine whether biomarkers of clot formation (products of von Willebrand Factor formation and activation) and clot resolution (product of fibrin degeneration) during COPD exacerbation predicted major cardiovascular events (MACE). The cohort was based on clinical data and biobank plasma samples from a trial including patients admitted with an acute exacerbation of COPD (CORTICO-COP). Neo-epitope biomarkers of formation and the activation of von Willebrand factor (VWF-N and V-WFA, respectively) and cross-linked fibrin degradation (X-FIB) were assessed using ELISAs in EDTA plasma at the time of acute admission, and analyzed for time-to-first MACE within 36 months, using multivariable Cox proportional hazards models. In total, 299/318 participants had samples available for analysis. The risk of MACE for patients in the upper quartile of each biomarker versus the lower quartile was: X-FIB: HR 0.98 (95% CI 0.65–1.48), VWF-N: HR 1.56 (95% CI 1.07–2.27), and VWF-A: HR 0.78 (95% CI 0.52–1.16). Thus, in COPD patients with an acute exacerbation, VWF-N was associated with future MACE and warrants further studies in a larger population.

INFLUENCE OF FIBRIN D AND DD FRAGMENTS ON FIBRINOGEN AND FIBRINOGEN FRAGMENT X POLYMERIZATION INITIATED BY THROMBIN OR ANCISTRON

Aim. Study of the role of the complex between the αC region and the BβN domain in the initial stages of fibrin polymerization has been investigated. Materials and Methods. Method of turbidimetry to study the influence of fibrinogen fragments D and DD on the polymerization and methods of isolation, purification, fragmentation for fibrinogen, monomer and cross-linked fibrin, fibrinogen X-fragment, Glu -plasminogen were used. Results. It was shown that fragment DD completely inhibited polymerization process in all the systems examined (“Fg + Thr”, “Fg + Anc H”, “X + Thr”, “X + Anc H”). Fragment D inhibited fibrin polymerization at all stages in the system “Fg + Thr”, but in the system “Fg + Anc H” it almost did not influence fibrin polymerization. In the both systems “X + Thr” and “X + Anc H” fragment D weakly inhibited the self-assembly of fibrin molecules into protofibrils, but accelerated the process of lateral association in the second system. Conclusions. The data obtained indicated that the complex between the αC region and the BβN domain of fibrin desA, on the initial stage of polymerization supported the rate of self-assembling and lateral association of fibrin desA protofibrils, protecting the oligomers against the depolymerizing influence of fibrinogen.

Full Characterization of Thrombotic Events in All Hospitalized COVID-19 Patients in a Spanish Tertiary Hospital during the First 18 Months of the Pandemic.

The presence of a procoagulant state, COVID-19-related coagulopathy, and an increased rate of thrombotic events (TEs) is widely known about. However, descriptive studies are scarce. Here, we conducted a large retrospective study including 2894 hospitalized COVID-19 patients followed up during the first 18 months of the pandemic to completely characterize any TE. Major TEs showed a 3.45% incidence rate. TEs were associated with increased intubation/90-day mortality risk [OR = 1.71, 95% CI (1.12–2.61), p < 0.013]. Venous thrombotic events (VTEs) were more frequent than arterial thrombotic events (ATEs) (72% vs. 28%), associated with enhanced levels of D-dimer (cross-linked fibrin derivatives formed during thrombolysis), which were related to mortality but more useful for early detection of thrombosis. In this regard, D-dimer plasma levels above 2014 µg/mL at hospital admission identify TEs with 91% accuracy (AUC = 0.91, p < 0.001), rising to almost 95% (AUC = 0.94, p < 0.001) with a cut-off value of 2666 µg/mL in VTEs. Moreover, 41% of TEs occurred in patients receiving LMWH thromboprophylactic treatments in hospital or domiciliary therapies. SARS-CoV-2 infection along with a sedentary lifestyle derived from the confinement in 2020 could be more determinant than a procoagulant state in patients with risk factors for TEs. Furthermore, the normal results obtained from the thrombophilia study after the acute process are linked to this independent procoagulant state and to SARS-CoV-2-derived coagulopathy.

Ethacrynic acid is an inhibitor of human factor XIIIa

BackgroundEthacrynic acid (EA) is a loop diuretic that is approved orally and parenterally to manage edema-associated diseases. Nevertheless, it was earlier reported that it is also associated with bleeding upon its parenteral administration. In this report, we investigated the effects of EA on human factor XIIIa (FXIIIa) of the coagulation process using a variety of techniques.MethodsA series of biochemical and computational methods have been used in this study. The potency and efficacy of human FXIIIa inhibition by EA was evaluated using a bisubstrate-based fluorescence trans-glutamination assay under near physiological conditions. To establish the physiological relevance of FXIIIa inhibition by EA, the effect on FXIIIa-mediated polymerization of fibrin(ogen) as well as the formation of fibrin(ogen) – α2-antiplasmin complex was evaluated using SDS-PAGE experiments. The selectivity profile of EA against other coagulation proteins was assessed by evaluating EA’s effect on human clotting times in the activated partial thromboplastin time (APTT) and the prothrombin time (PT) assays. We also used molecular modeling studies to put forward a putative binding mode for EA in the active site of FXIIIa. Results involving EA were the average of at least three experiments and the standard error ± 1 was provided. In determining the inhibition parameters, we used non-linear regression analysis.ResultsFXIIIa is a transglutaminase that works at the end of the coagulation process to form an insoluble, rigid, and cross-linked fibrin rich blood clot. In fact, inhibition of FXIIIa-mediated biological processes has been reported to result in a bleeding diathesis. Inhibition of FXIIIa by EA was investigated given the nucleophilic nature of the thiol-containing active site of the enzyme and the Michael acceptor-based electrophilicity of EA. In a bisubstrate-based fluorescence trans-glutamination assay, EA inhibited FXIIIa with a moderate potency (IC50 ~ 105 µM) and efficacy (∆Y ~ 66%). In SDS-PAGE experiments, EA appears to significantly inhibit the FXIIIa-mediated polymerization of fibrin(ogen) as well as the formation of fibrin(ogen) – α2-antiplasmin complex which indicates that EA affects the physiological functions of FXIIIa. Interestingly, EA did not affect the clotting times of human plasma in the APTT and the PT assays at the highest concentration tested of 2.5 mM suggesting the lack of effects on the coagulation serine proteases and potentially the functional selectivity of EA with respect to the clotting process. Molecular modeling studies demonstrated that the Michael acceptor of EA forms a covalent bond with catalytic residue of Cys314 in the active site of FXIIIa.ConclusionsOverall, our studies indicate that EA inhibits the physiological function of human FXIIIa in vitro which may potentially contribute to the bleeding complications that were reported with the association of the parenteral administration of EA.

Abstract 460: Covid-19 And Acute Limb Ischemia: Arterial Thromboemboli Composition And Architecture

Objectives: The incidence of acute limb ischemia (ALI) has increased since the emergence of coronavirus disease 2019 (COVID-19) and has been linked to the serum level of cross-linked fibrin degradation products (D-dimer). The purpose of this study is to understand arterial thromboembolic architecture in patients with ALI and how this is altered by COVID-19 infection. Methods: Arterial thromboembolic specimens were prospectively collected at a single institution, stored in an IRB-approved tissue bank at -80°C for preservation of architecture. Liquid chromatography mass spectrometry (LC-MS/MS) was used to identify in-vivo Factor XIII (FXIII) generated cross-links, critical determinants of clot stability and strength. Protein composition of thromboemboli was compared between COVID and non-COVID patients. Results: Arterial thromboemboli were obtained from four patients. Patient #1 had COVID pneumonia and a D-dimer of 14,690 ng/mL FEU and developed ALI secondary to embolization of an aortic thrombus. Patients #2-4 did not have COVID and developed ALI secondary to synthetic graft thrombosis, septic embolization from endocarditis, and embolization during aortic arch replacement. COVID thromboemboli demonstrated a higher abundance of immune response proteins compared to non-COVID thromboemboli. Eighty-one cross-links were identified across the four specimens (Fig 1). A six-fold greater degree of Fibrinogen Alpha Chain (FibA-FibA) cross-linking was present in the COVID specimen (Mean: 295,100) compared to the mean of the three non-COVID specimens (Mean: 52,973) (Figure 2). Conclusions: A higher degree of cross-linking and increased levels of innate and adaptive immune response proteins appear to be features of arterial thromboemboli in COVID-induced ALI. This supports a virus-associated hypercoagulable state in COVID-19 patients with ALI.

Clinical Confusion Concerning Increased D-Dimer Value during Pregnancy

Plasma D-dimer, a special cross-linked fibrin derivative, is produced when fibrin is degraded by plasminase. During pregnancy, D-dimer increases along with the increase of gestational age, and the reference value of plasma D-dimer (≤0.5 mg/L) traditionally used for the screening of venous thrombosis in the normal population is not applicable to the pregnant population. Due to the lack of uniform D-dimer detection methods or measurement units, there is currently no unified D-dimer reference values for pregnancy or puerperium. Each region or laboratory should establish its own pregnancy D-dimer reference value for different gestational weeks through blood coagulation function testing of large numbers of samples of different gestational periods. More and more studies have been conducted to investigate the association between D-dimer and venous thromboembolism (VTE) during pregnancy, gestational hypertensive disorders (GHD) and pregnancy outcome. We reviewed, herein, the generation and measurement of D-dimer, the reference values of D-dimer during normal pregnancy, and the association between D-dimer and some pathological pregnancies, intending to help clinicians develop a more thorough understanding of D-dimer during pregnancy.

Associations between hemostatic markers and mortality in COVID-19 – Compounding effects of D-dimer, antithrombin and PAP complex

In this single-center cohort study, we applied a panel of laboratory markers to characterize hemostatic function in 217 consecutive patients that underwent testing for COVID-19 as they were admitted to Linköping University Hospital between April and June 2020. In the 96 patients that tested positive for SARS-CoV-2 (COVID-19+), the cumulative incidences of death and venous thromboembolism were 24.0% and 19.8% as compared to 12.4% (p = 0.031) and 11.6% (p = 0.13) in the 121 patients that tested negative (COVID-19−). In COVID-19+ patients, we found pronounced increases in plasma levels of von Willebrand factor (vWF) and fibrinogen. Excess mortality was observed in COVID-19+ patients with the following aberrations in hemostatic markers: high D-dimer, low antithrombin or low plasmin-antiplasmin complex (PAP) formation, with Odds Ratios (OR) for death of 4.7 (95% confidence interval (CI95) 1.7–12.9; p = 0.003) for D-dimer >0.5 mg/L, 5.9 (CI95 1.8–19.7; p = 0.004) for antithrombin (AT) ˂0.85 kIU/l and 4.9 (CI95 1.3–18.3; p = 0.019) for PAP < 1000 μg/L. Compounding increases in mortality was observed in COVID-19+ patients with combined defects in markers of fibrinolysis and coagulation, with ORs for death of 15.7 (CI95 4.3–57; p < 0.001) for patients with PAP <1000 μg/L and D-dimer >0.5 mg/L and 15.5 (CI95 2.8–87, p = 0.002) for patients with PAP <1000 μg/L and AT ˂0.85 kIU/L. We observed an elevated fraction of incompletely degraded D-dimer fragments in COVID-19+ patients with low PAP, indicating impaired fibrinolytic breakdown of cross-linked fibrin.

Circulating D-Dimers Increase the Risk of Mortality and Venous Thromboembolism in Patients With Lung Cancer: A Systematic Analysis Combined With External Validation.

BackgroundD-dimer is a fibrin-degrading substance that is soluble and whose degradation is produced by plasma protein-mediated degradation of cross-linked fibrin. Previous investigations have shown a link between D-dimer and the mortality in lung cancer patients. However, different investigations varied whether D-dimer could predict prognosis in these patients.MethodsA meta-analysis and systematic review of all available cohort studies were performed on the link between circulating D-dimer levels and survival of lung cancer patients. Relevant studies were searched in Embase, Cochrane Library, and PubMed databases. Data from 540 lung cancer patients from the First Hospital of Soochow University and Sichuan Cancer Hospital were used for external validation.ResultsWe finally obtained 19 eligible cohort studies with pooled HR showing that high D-dimer levels contribute to death in tumor group (HR 1.62, 95% CI: 1.39–1.88, I2 = 75.0%). Further stratified analysis showed that higher circulating D-dimer in the advanced lung cancer group was linked to a 1.91-fold risk (HR = 2.91, 95% CI: 2.24–3.78, I2 = 6.0%). Incorporation of other variables, including days of follow-up, country, design, public year, population, disease status, and quality score, into the meta-regression model, indicated that disease status was an additional source of heterogeneity (p < 0.001). External validation of 540 patients also showed that high levels of D-dimer showed a higher risk of overall mortality (HR 1.39, 95% CI: 1.13–1.72, p = 0.002) and VTE events (HR 3.98, 95% CI: 1.99–8.70, p = 0.002) in lung cancer patients.ConclusionsHigh circulating plasma D-dimer levels independently predict long-term prognosis and the risk of venous thromboembolism in lung cancer.

Factor XIII is a newly identified binding partner for platelet collagen receptor GPVI‐dimer—An interaction that may modulate fibrin crosslinking

BackgroundIn the fibrin‐forming process, thrombin cleaves fibrinogen to fibrin, which form fibrils and then fibers, producing a gel‐like clot. Thrombin also activates coagulation factor XIII (FXIII), which crosslinks fibrin γ‐chains and α‐chains, stabilizing the clot. Many proteins bind to fibrin, including FXIII, an established regulation of clot structure, and platelet glycoprotein VI (GPVI), whose contribution to clot function is largely unknown. FXIII is present in plasma, but the abundant FXIII in platelet cytosol becomes exposed to the surface of strongly activated platelets. ObjectivesWe determined if GPVI interacts with FXIII and how this might modulate clot formation. MethodsWe measured interactions between recombinant proteins of the GPVI extracellular domain: GPVI‐dimer (GPVI‐Fc2) or monomer (GPVIex) and FXIII proteins (nonactivated and thrombin‐activated FXIII, FXIII subunits A and B) by ELISA. Binding to fibrin clots and fibrin γ‐chain crosslinking were analyzed by immunoblotting. ResultsGPVI‐dimer, but not GPVI‐monomer, bound to FXIII. GPVI‐dimer selectively bound to the FXIII A‐subunit, but not to the B‐subunit, an interaction that was decreased or abrogated by the GPVI‐dimer–specific antibody mFab‐F. The GPVI‐dimer–FXIII interaction decreased the extent of γ‐chain crosslinking, indicating a role in the regulation of clot formation. ConclusionsThis is the first report of the specific interaction between GPVI‐dimer and the A‐subunit of FXIII, as determined in an in vitro system with defined components. GPVI‐dimer–FXIII binding was inhibitory toward FXIII‐catalyzed crosslinking of fibrin γ‐chains in fibrin clots. This raises the possibility that GPVI‐dimer may negatively modulate fibrin crosslinking induced by FXIII, lessening clot stability.

Congenital and acquired disorders of secondary hemostasis

: Hemostasis depends on a large number of interdependent processes to prevent spontaneous bleeding and to stop hemorrhage when vascular injury occurs. Although it is commonly divided into primary and secondary for the purpose of simplification and patient evaluation, both processes occur almost simultaneously in vivo. The goal of secondary hemostasis is to produce a firm, cross-linked fibrin clot to stabilize the platelet plug. Fibrin is generated from a series of enzymatic reactions that comprise the coagulation cascade. In this review, we describe the background, physiology, diagnosis, and treatment of congenital and acquired coagulation factor deficiencies. For each condition, we discuss the appropriate laboratory assays as well as the most current treatment modalities. In addition, we describe in detail the oral and parenteral anticoagulants currently available in the United States, their mechanism of action, laboratory monitoring (if appropriate), and how to reverse their effect in case of hemorrhage or need for an emergent procedure. Finally, we review the effect of systemic conditions such as liver disease and disseminated intravascular coagulation (DIC) on the coagulation cascade.

Overall Survival Prediction of Advanced Cancer Patients by Selection of the Most Significant Baseline Serum Biomarker Combination.

Introduction: Consistent association between elevated baseline serum values and C-reactive protein (CRP), cross-linked fibrin degradation products (D-dimer), lactate dehydrogenase (LDH), decreased baseline serum albumin, absolute lymphocyte count to absolute monocyte count ratio (LMR), elevated absolute neutrophil count to absolute lymphocyte count ratio (NLR), elevated platelet count to absolute lymphocyte count ratio (PLR), and between some combinations of these biomarkers and the short overall survival of patients with malignant diseases has already been reported. These biomarkers are independent prognostic factors for cancer. Here, the most significant biomarker combination of these values was searched and studied in real-life advanced cancer patients of a single center. Methods: The authors retrospectively analyzed the association of the aforementioned biomarkers and their combination and OS of 75 consecutive cancer patients with locally advanced, recurrent, or metastatic diseases. Validated cut-off determination was used. Results: CRP, albumin, and PLR showed marked association with OS. Cut-off values for significant shorter OS were 30.65 mg/L (p < 0.001), 44.35 g/L (p < 0.001), and 168.20 (p < 0.001), respectively. Based on assessed biomarker cut-offs, four patient groups were created to determine whether biomarker values were out of range (ORV) compared to cut-off: 1) No ORV biomarkers (n = 24; OS = 26.07 months); 2) one ORV biomarker (n = 21; OS = 13.50 months); 3) two ORV biomarkers (n = 20; OS = 7.97 months), and 4) three ORV biomarkers (n = 10; OS = 3.91 months). Significant differences in OS were detected between the groups: For 1. vs. 2. hazard ratio (HR) = 3.0 (95% CI: 1.5–6.2), p = 0.003; for 1. vs. 3. HR = 4.1 (95% CI: 2.0–8.3), p < 0.001; and for 1. vs. 4. HR = 10.2 (95% CI: 4.2–24.6), p < 0.001. Conclusion: Based on our analysis, we can confirm that the complex monitoring of CRP, albumin, and PLR would provide a good estimation of OS. Large scale prospective studies are warranted to explore this and other useful combinations of prognostic biomarkers and their relationship to the well-established prognostic systems in real-life.

D-dimer - uloga u hemostaznom sistemu i primena u specifičnim kliničkim stanjima

D-dimer is a soluble fibrin degradation product, which is formed by the breakdown of cross-linked fibrin monomers, previously created under thrombin activity, formed as a result of hemostatic activation. The degradation of cross-linked fibrin occurs as part of the activation of the fibrinolysis system, whose task is to remove the created coagulum and ensure the patency of the blood vessel. This process takes place under the influence of the most important fibrinolytic enzyme, plasmin. D-dimer can, therefore be considered a biomarker of coagulation activation and fibrinolysis, and is routinely used to rule out venous thromboembolism (VTE). D-dimer is increasingly used to assess the risk of VTE recurrence and to help define the optimal duration of anticoagulant therapy in the treatment of patients with VTE, to diagnose disseminated intravascular coagulation (DIC), to screen medical patients at increased risk of VTE. In particular, the use of D-dimer, which is sometimes unjustifiably high, should be emphasized, as part of the monitoring and treatment of patients with COVID-19 infection. This review article aims to: (1) define the origin and role of D-dimer in the hemostatic system, (2) the method of performing the analysis, the selection of adequate tests and interpretation in accordance with the consideration of possible preanalytical variables that affect the measurement of D-dimer; (3) consideration of its application in specific clinical settings or diseases.

Oral therapy of recombinant Subtilisin QK-2 potentiates thrombolytic effect in a carrageenan-induced thrombosis animal model

• rSubtilisin QK has been shown to be resistant against pancreatic enzyme mimics. • rSubtilisin QK can be transferred into the bloodstream and all organs via intestinal absorption. • D-dimer and FDP levels elevated following rSubtilisin QK oral administration. • rSubtilisin QK has thrombolytic effects by oral gavage in vivo . • t-PA expression was increased by rSubtilisin QK, while PAI-1 showed the opposite trend. Thrombosis is a severe health care issue and one of the leading causes of death globally. Since presently used drugs have a number of limitations, safe and effective thrombolytic agents are still needed. Subtilisin QK, widely used as a component of functional food, presents high direct cross-linked fibrin degradation, without activation of plasminogen. It could be absorbed throughout intestine and infiltrate into all organs with fibrinolytic activity against the pancreatic enzyme and acidic pH mimicking the stomach in a time-dependent manner. Oral intake of rSubtilisin QK also elevated levels of fibrin/fibrinogen degradation products, D-dimer, and tissue plasminogen activator (t-PA) in plasma, and alleviated ear- and tail-thrombosis in κ-carrageenan-induced model. Furthermore, the levels of t-PA were increased in cultured HUVECs stimulated with rSubtilisin QK, while plasminogen activator inhibitor-1 showed the opposite trend simultaneously, suggesting the activation of plasminogen. In conclusion, rSubtilisin QK exerted thrombolytic effects in vivo and may be a potential oral thrombolytic agent.

Plasma levels of leucocyte elastase-generated cross linked fibrin degradation products (E-XDP) are elevated in chronic venous disease.

Patients with chronic venous disease (CVD) have elevated levels of leucocyte elastase (LE) released from the activation of leucocytes. In acute deep venous thrombosis (DVT), LE can degrade fibrin from the thrombus resulting in cross-linked fibrin degradation products (E-XDP) being released into the bloodstream. In patients with CVD the levels and significance of circulating E-XDP are unknown. We aimed to investigate the association between plasma E-XDP concentration and severity of CVD. Levels of E-XDP were quantified with a specific enzyme-linked immunosorbent assay (ELISA) in plasma from 142 consecutively recruited CVD patients (mean age 64 years, (range 23–89), 81 were females and 61 males). Patients were also divided into three groups based on CVD severity using the C-class of the Clinical-Etiological-Anatomical-Pathophysiological (CEAP) classification, with C 0–1 class as the reference group, C 2–3 as the second group and C 4–6 as the third group with the most severely affected patients. We found significantly elevated levels of E-XDP in patients with C 4–6 compared with patients with C 0–1 (p = 0.007) and increased with increasing disease severity across the groups (p = 0.02). Significant independent association was observed between levels of E-XDP and the classes C 4–6 after adjustment for age and sex (p < 0.05), but the association was no longer significant after further adjustment for use of statins, use of anticoagulants and history of DVT (p = 0.247). This exploratory study shows that E-XDP levels are elevated in patients with CVD, encouraging further studies on the role of E-XDP in CVD.

Novel venous thromboembolism mouse model to evaluate the role of complete and partial factor XIII deficiency in pulmonary embolism risk

BackgroundVenous thrombosis (VT) and pulmonary embolism (PE), collectively venous thromboembolism (VTE), cause high mortality and morbidity. Factor XIII (FXIII) crosslinks fibrin to enhance thrombus stability and consequently may influence PE risk. Elucidating mechanisms contributing to PE is limited by a lack of models that recapitulate human PE characteristics. ObjectiveWe aimed to develop a mouse model that permits embolization of red blood cell (RBC)‐ and fibrin‐rich VT and determine the contribution of FXIII to PE risk. Methods and ResultsIn a thrombin‐infusion PE model, F13a+/+, F13a+/−, and F13a−/− mice had similar incidence of microthrombi in the lungs; however, thrombi were small, with low RBC content (≤7%), unlike human PEs (~70%). To identify a model producing PE consistent with histological characteristics of human PE, we compared mouse femoral vein electrolytic injury, femoral vein FeCl3 injury, and infrarenal vena cava (IVC) stasis models of VT. Electrolytic and FeCl3 models produced small thrombi with few RBCs (5% and 4%, respectively), whereas IVC stasis produced large thrombi with higher RBC content (68%) that was similar to human PEs. After IVC stasis and ligature removal (de‐ligation) to permit thrombus embolization, compared to F13a+/+ mice, F13a+/− and F13a−/− mice had similar and increased PE incidence, respectively. ConclusionsCompared to thrombin infusion‐, electrolytic injury‐, and FeCl3‐based models, IVC stasis produces thrombi that are more histologically similar to human thrombi. IVC stasis followed by de‐ligation permits embolization of existing RBC‐ and fibrin‐rich thrombi. Complete FXIII deficiency increases PE incidence, but partial deficiency does not.

The role of serum D-dimer in the diagnosis of periprosthetic joint infection and timing of reimplantation.

D-dimer is a fibrin degradation product formed by the destruction of the cross-linked fibrin clot by activation of the coagulation system. Many studies have shown that systemic and local infections cause fibrinolytic activities. The purpose of this study was to determine the role of serum D-dimer in the diagnosis of periprosthetic joint infection (PJI) and the timing of reimplantation. The patients who underwent primary and revision knee or hip arthroplasty between July 2018 and May 2019 were prospectively evaluated. All surgeries were performed by the same surgeon. 60 of 71 patients who met our criteria were included in the study, comprising of 27 primary arthroplasties (PA), 21 two-stage septic revision (SR) and 12 aseptic revision (AR). We analyzed D-dimer, C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) levels. The values of the septic group were evaluated as two groups; Group 1 (before the first stage) and Group 2 (before the second stage) to investigate the role of D-dimer in determining the timing of reimplantation. D-dimer level was significantly higher in the SR group compared to PA and AR groups (p<0.001 and p=0.045). CRP and ESR levels were significantly higher in the SR group compared to PA and AR groups. There was no statistically significant difference between preoperative D-dimer levels of Group 1 and Group 2 (p=0.2). Serum D-dimer level can be used as an easy and inexpensive test to support the diagnosis of PJI. But the D-dimer level is not useful to determine the timing of reimplantation.

Correlation of plasma D-dimer levels with breast carcinoma

Background: Breast cancer is the most commonly occurring cancer in women. There is a correlation between cancer and hyper coagulation. Carcinoma increases the level of cross-linked fibrin degradation product (plasma D-dimer), indicative of systemic activation of fibrinolysis, hemostasis and angiogenesis. This study attempted to correlate raised plasma D-dimer in breast cancer.Methods: A retrospective study was conducted in the department of general surgery in GCS hospital, Ahmedabad where in patients admitted and treated for breast cancer between July 2020 to June 2021 were selected. Plasma D-dimer levels were correlated with final histopathological examination of breast specimen.Results: Plasma D-dimer levels were increased with advancing stage of disease, lymph node involvement and lymphovascular invasion. There was no significant relationship with increase in tumour size and histopathological grade of tumour.Conclusions: Plasma D-dimer levels are elevated in breast cancer, especially with advanced stage. It is an important marker of clinical stage, lymphovascular invasion, lymph node involvement. However it does not correlate with tumour size and histological grade. So pre- operative plasma D-dimer level is a safe, cost effective and convenient method for prediction of advanced stage in breast cancer.