Fibrinogen Fragments Research Articles

D-dimer and fibrinogen indicate ischemic risk in patients with atrial fibrillation after percutaneous coronary intervention

BackgroundThis study aimed to evaluate the association of antiphospholipid antibodies (aPL) and conventional markers of coagulation with ischemic and bleeding risk in patients with atrial fibrillation (AF) undergoing percutaneous coronary intervention (PCI).MethodsIn this prospective two-center observational cohort study, patients with AF and an indication for oral anticoagulation (OAC) were enrolled after PCI. Blood was drawn on day 1–3 after PCI. Dilute Russell’s viper venom time was used to determine lupus anticoagulant (LA) in OAC-free plasma. Anti-cardiolipin (aCL) IgG, IgM, and anti-β2-Glycoprotein 1 (aβ2GP1) IgG were analyzed by enzyme-linked immunosorbent assay (ELISA). Fibrinogen (FIB), d-dimer, and prothrombin fragment 1 and 2 (PF 1 + 2) were measured in citrated plasma. The primary ischemic outcome was time to major adverse cardiovascular events (MACE; death, myocardial infarction, or stroke) assessed at 6 months. Bleeding was defined according to International Society on Thrombosis and Haemostasis.Results158 patients were enrolled between May 2020 and May 2021 on day 1–3 after PCI. The median age was 78 years (interquartile range [IQR] 72–82), 111 (70%) were male, and 39 (25%) presented with acute coronary syndrome. D-dimer was elevated in 74 (47%) patients, FIB was increased in 40 (25%) and PF1 + 2 in 68 (43%) patients. 32 (20%) patients had ≥ 1 antiphospholipid antibody elevated (aPL; LA: 19 [12%], aCL: 14 [9%], aβ2GP1: 2 [1%]). The presence of aPL was neither significantly associated with MACE (HR 1.46, 95% CI [0.39–5.49], p = 0.579), nor bleeding (HR 1.07 [0.30–3.84], p = 0.917). Elevated d-dimer was significantly associated with higher risk for MACE (HR 5.06 [1.09–23.41], p = 0.038) and major bleeding (HR 7.04 [1.58–31.47], p = 0.011). Elevated D-dimer increased the predictive capacity of HAS-BLED for major bleedings (HAS-BLED: AUC 0.71 [0.60–0.83] vs. HAS-BLED + d-dimer: AUC 0.79 [0.70–0.88]; p = 0.025). Increased levels of FIB were associated with higher risk for MACE (HR 3.65 [1.11–11.96], p = 0.033).ConclusionBiomarkers of coagulation might be suitable to assess ischemic and bleeding risk in patients with AF following PCI.

Fibrinogen Cleavage by Lectin Pathway Proteases: Roles and Implications

The lectin pathway proteases (MASPs) are diverse in regard to their substrate selectivity and also have substrates outside the complement pathway. These proteases have been reported to cleave proteins of the coagulation cascade. MASP-1 has thrombin-like activity and therefore cleaves fibrinogen, fibrin and prothrombin. MASP-2 also participates in coagulation by cleaving prothrombin. Thus, these proteases are key players in the process of thrombosis and thrombolysis, therefore have implications in thrombotic vascular diseases. The cleaved fragments of fibrinogen and fibrin, consequently produced during coagulation and fibrinolysis, are important in the repair process and hence may have involvement in inflammatory and fibro-proliferative diseases. Also, altered levels of MBL and its associated serine proteases reported to be involved in different diseases such as cardiovascular diseases, diabetes, cancer, sepsis, stroke, COVID-19 etc. may have a role in thrombosis and thrombolysis. Upregulated lectin pathway-associated proteins predispose towards autoimmune disease susceptibility and may also exhibit off-target activities resulting in clot formation. Thus, the lectin pathway and its associated proteases are of great importance in normal circumstances where it plays a role in the maintenance of hemostasis and homeostasis, playing a preventive role in infections. Modulation in the level of MASPs proteases could be developed as a promising approach to treat a variety of infections and diseases.

1265: EFFECTS OF ENTERAL PROTEASE INHIBITION ON COAGULATION PROTEOLYSIS AFTER TRAUMA-HEMORRHAGIC SHOCK

Introduction: Coagulopathy and uncontrolled proteolysis are frequent sequelae of circulatory shock, and are associated with poor outcomes. We have previously demonstrated that enteral protease inhibition improves hemodynamics and survival in experimental shock. However, the mechanisms of inappropriate systemic protease activity in shock are not well understood. Our aim was to identify the effects of enteral protease inhibition on plasma proteolysis by examination of the plasma peptidome in experimental trauma-hemorrhagic shock (T-HS). Methods: Anesthetized Wistar rats were subjected to experimental T-HS with laparotomy. An oral catheter was inserted into the proximal duodenum and either gabexate mesilate+Golytely (GM) (10mg/kg) (n=8) or Golytely as a vehicle (n=8) was enterally infused starting 30min into the hypotension period. Blood was removed to achieve a mean blood pressure (MAP) of 35-40mmHg for 90min before reperfusion with lactated Ringer’s to obtain a target MAP of ≥65mmHg. Blood was taken for mass spectrometry peptidomics analysis at baseline and 120min after reperfusion. Results: Plasma proteolysis was increased 8.8±0.9 fold in the Shock-control group, and was reduced to 2.0±0.2 times baseline values in GM-treated animals (p< 0.0001). Despite representing only 0.3% of all proteins found in the peptidome, fibrinogen fragments comprised 11.3% of all peptides recovered after T-HS in our model, and were often non-canonically cleaved. Enteral GM decreased proteolysis of other proteins involved in coagulation, including: C4 (increased 1092±131% from baseline in Shock-control vs 154±22% in GM-treated animals, p< 0.0001); Kininogen-1 (relative intensity 90.5±20.4 in Shock-control vs. 19.8±1.4 in GM-treated group, p< 0.0001); Protein C (118% in Shock-control vs. GM-treated animals, p=0.0012). In addition, fragments from inter-alpha-trypsin inhibitor were exclusively found in the Shock-control group, suggesting breakdown of anti-proteases in the blood after shock. Conclusions: Enteral protease inhibition with gabexate mesilate significantly reduced plasma proteolysis after experimental T/HS as measured by peptidomics, especially of proteins involved with coagulation. This approach opens new insights for the understanding and treatment of Trauma-hemorrhagic shock, particularly related to coagulopathies.

Thrombin Generation and D-Dimer for Prediction of Disease Progression and Mortality in Patients with Metastatic Gastrointestinal Cancer.

Simple SummaryAssessing the prognosis of a patient with cancer is of the greatest clinical interest and can provide useful information on the type and intensity of the anticancer treatment to be administered. The emerging predictive role of coagulation biomarkers in cancer prognosis needs to be confirmed by prospective cohort studies. In this study, we evaluated whether prechemotherapy levels of thrombotic biomarkers may predict for early disease progression and overall survival in a large prospective cohort of patients with metastatic gastrointestinal cancer specifically enrolled for the intended aims. We found that pretreatment thrombin generation and D-dimer appear to be promising candidate biomarkers for both outcomes.Background: the tight and reciprocal interaction between cancer and hemostasis has stimulated investigations on the possible role of hemostatic biomarkers in predicting specific cancer outcomes, such as disease progression (DP) and overall survival (OS). In a prospective cohort of newly diagnosed metastatic gastrointestinal (GI) cancer patients from the HYPERCAN study, we aimed to assess whether the hemostatic biomarker levels measured before starting any anticancer therapy may specifically predict for 6-months DP (6m-DP) and for 1-year OS (1y OS). Methods: plasma samples were collected and tested for thrombin generation (TG) as global hemostatic assay, and for D-dimer, fibrinogen, and prothrombin fragment 1 + 2 as hypercoagulation biomarkers. DP and mortality were monitored during follow-up. Results: A prospective cohort of 462 colorectal and 164 gastric cancer patients was available for analysis. After 6 months, DP occurred in 148 patients, providing a cumulative incidence of 24.8% (21.4–28.4). D-dimer and TG endogenous thrombin potential (ETP) were identified as independent risk factors for 6m-DP by multivariate Fine–Gray proportional hazard regression model corrected for age, cancer site, and >1 metastatic site. After 1 year, we observed an OS of 75.7% (71.9–79.0). Multivariate Cox regression analysis corrected for age, site of cancer, and performance status identified D-dimer and ETP as independent risk factors for 1y OS. Patients with one or both hemostatic parameters above the dichotomizing threshold were at higher risk for both 6m-DP and 1-year mortality. Conclusion.: in newly diagnosed metastatic GI cancer patients, pretreatment ETP and D-dimer appear promising candidate biomarkers for predicting 6m-DP and 1y OS. In this setting, for the first time, the role of TG as a prognostic biomarker emerges in a large prospective cohort.

Fibrinogenolysis in Venom-Induced Consumption Coagulopathy after Viperidae Snakebites: A Pilot Study.

Envenomations that are caused by Viperidae snakebites are mostly accompanied by venom-induced consumption coagulopathy (VICC) with defibrination. The clinical course of VICC is well described; however, reports about its detailed effects in the hemocoagulation systems of patients are sparse. In this pilot study, we prospectively analyzed the changes in plasma fibrinogen that were caused by the envenomation of six patients by five non-European Viperidae snakes. Western blot analysis was employed and fibrinogen fragments were visualized with the use of specific anti-human fibrinogen antibodies. All of the studied subjects experienced hypo- or afibrinogenemia. The western blot analysis demonstrated fibrinogenolysis of the fibrinogen chains in all of the cases. Fibrinogenolysis was considered to be a predominant cause of defibrination in Crotalus, Echis, and Macrovipera envenomation; while, in the cases of VICC that were caused by Atheris and Calloselasma envenomation, the splitting of the fibrinogen chains was present less significantly.

A novel ex vivo approach for measuring plasminogen activation upon established plasma clots

BackgroundThe fibrinolytic system plays a critical role in maintaining hemostasis. Central to fibrinolysis is the degradation of fibrin by plasmin, produced in the circulation following the activation of plasminogen by plasminogen activators (PAs). Accurately measuring the plasminogen activation rate is vital for the understanding of fibrinolytic processes, particularly in the context of thrombolysis. Yet, due to the insoluble nature of fibrin, in vitro and ex vivo investigations of PA‐mediated plasminogen activation have proven challenging. As researchers frequently adopt soluble fibrinogen fragments and/or alter the experimental system beyond what is physiologically relevant, they limit the validation and interpretation of their findings. Here, we present a novel, high‐throughput assay for measuring plasminogen activation rates on natural, plasma‐derived fibrin that optimally simulates in vivo conditions. MethodHuman plasma was used as the source of plasmin(ogen) and fibrin(ogen). “Halo‐shaped” plasma clots were produced in a 96‐well plate using a thrombin‐containing clotting mixture, ensuring that an optically compatible and plasma‐free center is maintained in each well. Subsequent additions of a plasmin chromogenic substrate and different PAs were followed by absorbance measurements over time to extract the corresponding enzyme kinetics information. Results and DiscussionValidation experiments demonstrated the capability of our approach to accurately model fibrin‐dependent and ‐independent plasminogen activation as well as sensitively detect variations in plasminogen and fibrinogen plasma levels. ConclusionThis assay allows a straightforward, yet powerful, measurement of plasminogen activation rates on established plasma clots, with the capability of properly assessing fibrin‐ and non–fibrin‐dependent plasminogen activation by various therapeutic PAs.

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.

PRFF Peptide Mimic Interferes with Toxic Fibrin-Aβ42 Interaction by Emulating the Aβ Binding Interface on Fibrinogen.

Cerebrovascular dysfunction is a common phenomenon in Alzheimer's patients, where fibrinogen is a major player. With the blood-brain barrier compromised, fibrinogen gains access to the brain, where its interaction with Aβ42 results in plasmin-resistant abnormal blood clots that are deposited in the cerebral blood vessels, a condition commonly encountered in Alzheimer's disease (AD) patients called cerebral amyloid angiopathy (CAA). So far, there have been no effective therapeutics available to combat AD-associated CAA. This study reports a 13-amino acid peptide (Pα-NPGRPEPGSAGTW) as a potential inhibitor of the fibrin-Aβ42 interaction along with the property to dissolve pre-existing plasmin-resistant abnormal clots. Strikingly, the identified sequence was found to be partially similar to a fragment of the fibrinogen α-chain reported to bind Aβ42, the plasmin-resistant fibrinogen fragment (PRFF). Mechanistically, Pα interacts with Aβ42 in place of fibrinogen, thus inhibiting the toxic fibrin-Aβ42 interaction. However, it does not interfere with normal fibrin polymerization.

Quantitative Structural Characterization of Fibrinogen's αC Regions in Fibrin Monomers and Oligomers

IntroductionMolecular structural characterization of fibrinogen and fibrin provide a basis for prophylaxis and treatments of thrombotic and hemostatic disorders. The flexible C-terminal parts of fibrinogen's Aα chains (αC regions) have been shown to play a role in fibrin formation, cross-linking by factor XIIIa, fibrinolysis, etc. The αC regions comprise about ¼ of the whole molecule by mass, but, because of their extreme flexibility, their structure is missing in the X-ray crystallography of fibrinogen and fibrin fragments. Here, we used high-resolution atomic force microscopy to examine the structure of the αC regions in fibrinogen monomers and in fibrin oligomers formed at the early stages of fibrin polymerization.MethodsThe surface of freshly cleaved graphite was rendered hydrophilic with an amphiphilic hydrocarbon-glycine modifier (Klinov et al., Nanotechnology, 2007, 18, 225102). The modified surface provides favorable conditions for protein absorption and allows obtaining high-resolution imaging of single protein molecules in air. To further improve the resolution, super sharp homemade cantilevers were also used.Plasma-purified full-length human fibrinogen and fibrinogen sub-fraction I-9 lacking most of the αC regions were imaged before and after thrombin treatments. As judged from SDS-PAGE, the I-9 fibrinogen lacked the C-terminal portions of its Aα chains and contained the Aα chain core remnants from 46.5 kDa to 22.6 kDa (or ~66% to 32% of the mass of intact Aα chain). As estimated from these measurements, the sub-fraction lacks virtually all of the αC-domains (Aα392-610) and variable lengths of the αC-connector (Aα221-391). The Bβ and γ chains of the I-9 fibrinogen were intact.ResultsWe analyzed the incidence of intact αC regions, their overall shape, and measured the contour length of the αC regions in the full-length and truncated (I-9) fibrinogen variants as well as their derivatives. In addition, we compared fibrin polymerization kinetics and the final clot structures. The incidence of the αC regions was 50% in the full-length fibrinogen compared to 19% in fibrinogen I-9. In the full-length fibrinogen, we could identify compact globular C-terminal αC-domains linked to the bulk molecule via flexible filamentous αC-connectors, confirming the previously proposed structure of the αC regions. The residual αC regions in fibrinogen I-9 were significantly shorter than in the full-length fibrinogen.To see if the structure of the αC regions changed after conversion to fibrin, we compared the structures of fibrin oligomers formed from the full-length and I-9 fibrinogen variants by thrombin. Up to 80% of the potentially existing αC regions were visualized and quantified in the full-length fibrin; they were highly heterogeneous in their length and configurations. Importantly, conversion of the full-length fibrinogen to fibrin was accompanied by an increase of the incidence and length of the αC regions as well as transitions from more compact conformations, such as a globule on a string, to extended and more flexible offshoots. On the other hand, in fibrinogen I-9 more αC regions were visualized after conversion to fibrin, but the contour length and morphology of shortened αC regions did not change upon fibrin oligomerization.Concurrent dynamic turbidimetry, confocal microscopy, and scanning electron microscopy revealed that trimming of the αC regions slowed down fibrin formation, which correlated with longer protofibrils, thinner fibers, and a denser network. No structural distinctions, except for the incidence of the αC regions, were revealed in laterally aggregated protofibrils made of the full-length or I-9 fibrinogens, suggesting a pure kinetic effect of the αC regions on the fibrin architecture.ConclusionsOur results confirmed that after conversion of fibrinogen to fibrin, the αC regions become exposed, undergo partial unfolding and/or conformational elongation. However, the effects of the αC regions on fibrin formation have a kinetic nature due to reduction of the threshold length of protofibrils, leading to accelerated lateral aggregation. The final structure and macroscopic properties of fibrin clots are affected by kinetics of the earliest stages of fibrin self-assembly. Therefore, these early polymerization steps comprise a good potential therapeutic target to modulate the structure and mechanical properties of blood clots. DisclosuresNo relevant conflicts of interest to declare.

Synthetic blood and blood products for combat casualty care and beyond.

Synthetic biology adopts an engineering design approach to create innovative treatments that are reliable, scalable, and customizable to individual patients. Interest in substitutes for allogenic blood components, primarily red blood cells and platelets, increased in the 1980s because of concerns over infectious disease transmission. However, only now, with emerging synthetic approaches, are such substitutes showing genuine promise. Affordable alternatives to donated blood would be of enormous benefit worldwide. Several approaches to replacing the oxygen-carrying function of red cells are under advanced investigation. Hemoglobin-based oxygen carriers incorporate modifications to reduce the renal toxicity and nitric oxide scavenging of free hemoglobin. While use of earlier-generation hemoglobin-based oxygen carriers may be limited to circumstances in which blood transfusion is not an option, recent advances in chemical modification of hemoglobin may eventually overcome such problems. Another approach encases hemoglobin molecules in biocompatible synthetic nanoparticles. An alternative is the ex vivo production of red cells in bioreactors, with or without genetic manipulation, that offers the potential of a universal donor product. Various strategies to manufacture synthetic platelets are also underway, ranging from simple phospholipid liposomes encapsulating adenosine diphosphate and decorated with fibrinogen fragments, to more complex capsules with multiple receptor peptide sequences. Ex vivo production of platelets in bioreactors is also possible including, for example, platelets derived from induced pluripotent stem cells that are differentiated into a megakaryocytic lineage. Prior to clinical use, trials assessing synthetic blood components must evaluate meaningful safety and effectiveness outcomes in relatively large numbers of critically ill patients. Overcoming these challenges may be as much a hurdle as product design. This article reviews the state of the science of the synthetic biology approach to developing blood component substitutes.

Патогенетические механизмы развития гипофибриногенемии при COVID-19

Введение. Коагулопатия у пациентов с новой коронавирусной инфекцией COVID-19 имеет важное значение в течении и прогнозе заболевания. Типичными проявлениями коагулопатии в острой фазе болезни является склонность к гиперкоагуляции, гиперфибриногенемии и повышенному тромбообразованию. Гипофибриногенемия при COVID‑19 широко не представлена в литературе, и частота, а также причины её развития неизвестны. Одной из возможных причин возникновения гипофибриногенемии является применение генно-инженерных биологических препаратов (ГИБП), однако механизмы развития данного состояния не ясны. Цель исследования: определить роль терапии антагонистами рецепторов интерлейкина-6 при развитии гипофибриногенемии у пациентов с COVID-19-пневмонией, клиническую значимость гипофибриногенемии и механизм её возникновения. Материалы и методы. В ретроспективное исследование путём случайной выборки включено 239 пациентов в возрасте от 18 до 96 лет с подтверждённой методом полимеразной цепной реакции SARS-CoV-2 инфекцией, КТ-признаками (при поступлении в стационар) вирусной пневмонии 2–4 степени и длительностью госпитализации более 2 сут. Всем пациентам проводили лабораторный мониторинг, включавший определение значений гемоглобина, тромбоцитов, С-реактивного белка (СРБ), печёночных ферментов и фибриногена по Клаусу. Всем пациентам проводилась стартовая терапия гидроксихлорохином и азитромицином. Тоцилизумаб на момент исследования был единственным ГИБП, применяемым в стационаре. Терапия тоцилизумабом была проведена 164 (68,6%) пациентам. В исследовании учитывали максимальные и минимальные значения гемоглобина, тромбоцитов, фибриногена и СРБ в течение всей госпитализации. Результаты. Гипофибриногенемия различной степени выраженности наблюдалась у 39 пациентов, получивших тоцилизумаб, что значимо выше частоты гипофибриногенемии у 2 пациентов, не получавших терапию тоцилизумабом (ОР = 8,9; 95% ДИ = 2,2–25,9). Заключение. Применение тоцилизумаба у пациентов с COVID‑19 достоверно повышает риск развития гипофибриногенемии. Background. Coagulopathy in patients with new coronavirus infection COVID-19 is important for the course and prognosis of the disease. Hypercoagulation, hyperfibrinogenemia and thrombosis are typical manifestations of coagulopathy in the acute phase of the disease. Hypofibrinogenemia in COVID‑19 is not widely reported in the literature, and the frequency and reasons of its development are unknown. The use of genetically engineered biological drugs (GEBD) may be one of the possible causes of hypofibrinogenemia but the mechanisms of its development are not clear. Objectives: to determine the role of therapy with interleukin-6 receptor antagonists in the development of hypofibrinogenemia in patients with COVID-19-pneumonia, the clinical significance of hypofibrinogenemia and the mechanism of its occurrence. Patients/Methods. A randomized retrospective study included 239 patients aged 18 to 96 years with confirmed by polymerase chain reaction SARS-CoV-2 infection, computed tomography signs (upon admission to the hospital) of viral pneumonia (grade 2–4) and a duration of hospitalization of more than 2 days. All patients underwent laboratory monitoring, including the determination of hemoglobin, platelets, C-reactive protein (CRP), liver enzymes and fibrinogen according to Claus. All patients received initial therapy with hydroxychloroquine and azithromycin. At the time of the study, tocilizumab was the only GEBD drug used in the hospital. Therapy with tocilizumab was performed in 164 (68.6%) patients. The study took into account the maximum and minimum values of hemoglobin, platelet, fibrinogen and CRP levels during the hospitalization. Results. Hypofibrinogenemia of varying severity was observed in 39 patients treated with tocilizumab, that was significantly higher than hypofibrinogenemia frequency in 2 patients who did not receive tocilizumab (RR = 8.9; 95% CI = 2.2–25.9). Conclusions. Tocilizumab usage in patients with COVID-19 significantly increases the risk of hypofibrinogenemia.

Abstract IA08: Incorporating proteomics in bladder cancer molecular diagnosis

Abstract Proteins are directly linked to cell phenotype, hence accrue interest as biomarkers and therapeutic targets. Investigating the urine proteome as a source of biomarkers entails multiple technical challenges, with the main being the protein concentration vast dynamic range and presence of proteins in multiple modified forms. As a result, protein enrichment strategies in combination to different mass spectrometry platforms are regularly employed as main discovery tools, providing complementary experimental outputs. Verification and further biomarker validation regularly involve application of ELISA and, more recently, multiplex mass spectrometry (MS)-based assays (Multiple Reaction Monitoring–MS). In the course of this presentation, an overview of urine proteomics, as applied in bladder cancer research, will be provided. In brief, using the armamentarium of existing methodologic approaches, multiple urinary proteins have been associated with bladder cancer phenotypes and/or attributed prognostic or predictive value, including the Nuclear Matrix Protein 22 (NMP22) and Bladder Tumor Antigen (BTA; corresponding to complement factor H-related protein), approved by the FDA for use in disease diagnosis and/or surveillance. Additional proteins frequently reported in the literature as associated with bladder cancer detection and/or staging include various forms of apolipoproteins, proteins of the extracellular matrix (such as collagen and fibrinogen fragments), matrix metalloproteinases, cytokeratin fragments as well as proteins involved in apoptosis (such as survivin) or inflammation (including multiple cytokines). Reports also support potential of some of these proteins when evaluated in the form of biomarker panels, as predictors of BCG response in high-risk non-muscle invasive bladder cancer. Along these lines, simultaneous analysis of multiple markers through ELISA or mass spectrometry-based platforms has provided increased accuracies in comparison to single biomarkers. Hematuria is a frequent confounding variable in urinary protein measurements, rendering establishment of signal specificity via optimized ELISA assays or mass spectrometry platforms a current technological target. Direct links of urinary protein changes to bladder tissue events have been frequently reported (such as the cases of profilin 1 or activated leukocyte cell adhesion molecule). Even though disproportionate to the numerous reported findings (frequently compromised by suboptimal experimental design), collectively, promising urinary protein findings in association to bladder cancer phenotypes and contexts of use are existent, awaiting proper validation in large-scale prospective trials. Main research paths that now open up include: refinement of existing bladder cancer molecular subtypes, with the addition of information at the protein level, and placing urinary markers in this context; and, in parallel, design and running of proper validation studies as well as clinical sample sharing, to define optimal marker combinations per clinically relevant bladder cancer context of use. In the latter, special emphasis should be placed on prediction of treatment response, in order to maximize benefit from available treatment modalities. Citation Format: Antonia Vlahou. Incorporating proteomics in bladder cancer molecular diagnosis [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2019 May 18-21; Denver, CO. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(15_Suppl):Abstract nr IA08.

Dominant Role of αIIbβ3 in Platelet Interactions with Polymerizing Cross-Linked Fibrin

The formation of a stable platelet thrombus in vivo is accompanied by the generation of thrombin and the conversion of fibrinogen to cross-linked fibrin. This requires thrombin's action on fibrinogen, producing fibrin monomers, and factor XIII, catalyzing the reciprocal transamidation of the C-terminal γ-chain peptides from adjacent fibrinogen molecules. While the interaction of platelets with fibrinogen has been shown to be mediated by the interaction of fibrinogen γ-chain residues 404-411 with the RGD pocket on integrin αIIbβ3, the interaction with cross-linked fibrin is less well understood despite its clinical relevance. We previously reported that activated, but not unactivated, αIIbβ3 can support binding to D-dimer, the cross-linked plasmin fragment of fibrin, and 'D98' a fibrinogen fragment lacking the γ404-411 sequence. Here we studied the interaction of washed platelets in buffer containing 1 mM Mg2+ with polymerizing fibrin in an aggregometer by adding 0.2 U/ml thrombin and monitoring light transmission. To prevent the interaction of γ12 with the RGD pocket, studies were performed in the presence of 150 μM RGDW, a concentration that completely eliminates aggregation of the platelets to a thrombin receptor activating peptide (T6). RGDW eliminated the initial wave of platelet aggregation induced by thrombin, but did not prevent the appearance of a delayed wave (DW), which appeared after ~4-5 min (Panel A-B). Parallel immunoblot studies demonstrated that cross-linking of fibrinogen γ-chains, (γ-γ dimers) appeared within 1 min after adding thrombin and was ~85% complete by 5 min (Panel C). Thus, both cross-linked and uncross-linked fibrin(ogen) was present during the DW. Adding fibrinogen at 10-100 μg/ml enhanced the amplitude of the DW in a dose-dependent manner (Panel D). The fibrin polymerization inhibitor Gly-Pro-Arg-Pro (GPRP; 5 mM) dramatically inhibited the amplitude of the DW, supporting the importance of fibrin polymerization (Panel B). An antibody to platelet receptor α2β1 (6F1) had no effect on the slope or amplitude of the DW, whereas an antibody to GPIb (6D1) had a mild effect on the slope but not the amplitude of the DW. Since GPVI has variably been reported to interact with fibrin, we tested whether there was evidence of GPVI-mediated signaling during the DW. The known GPVI ligands/activators collagen and collagen-related peptide (CRP) (controls), produced marked increases 3 min after the onset of aggregation in phosphorylation of PLC-γ2-Y759, Syk-Y525/526, and LAT-Y191. Thrombin alone caused a major increase in phosphorylation of Syk-Y525/526 and a slight increase in phosphorylation of PLCγ2-Y759; however, adding RGDW dramatically inhibited the phosphorylation of Syk. We conclude that at 3 min after the onset of the DW there is minimal or no GPVI-mediated signal transduction. Similarly, neither the Src inhibitor PP2 at 100 μM or the Syk inhibitor VI at 1 μM, both of which inhibit CRP-induced platelet aggregation, inhibited the DW. Since polymerizing fibrin has the potential for multiple interactions with different platelet αIIbβ3 receptors, thus increasing the avidity, we compared the ability of αIIbβ3 antagonists to inhibit T6-induced aggregation versus the DW of polymerizing fibrin. Whereas the γ400-411 peptide was able to nearly completely inhibit T6-induced platelet aggregation at ~150 μM, it had virtually no impact on the DW at concentrations up to 500 μM. RGDW eliminated T6-induced aggregation at ~5 μM, but required 100-fold as much to produce ~80% inhibition of the DW. Similarly, eptifibatide was able to nearly eliminate T6-induced aggregation at ~5 μM, but 100-fold as much was required to eliminate the DW. Thus, even if αIIbβ3 antagonists do not inhibit the DW at doses that eliminate T6-induced aggregation, one cannot infer that other receptors are involved. Consistent with a dominant role for activated αIIbβ3 in the process, 10 mM EDTA essentially eliminated the DW and reptilase-induced fibrin polymerization did not lead to platelet incorporation unless the platelets were activated with T6 or another αIIbβ3 activator. In addition, both D-dimer and D98 inhibited the thrombin-induced DW at 200 µg/ml. We conclude that activated αIIbβ3 plays a dominant role in supporting high avidity platelet interactions with polymerizing, partially cross-linked, fibrin, and that the mechanism has features akin to, but distinct from the interaction of αIIbβ3 with fibrinogen. Figure Disclosures Coller: CeleCor: Consultancy, Equity Ownership, Research Funding; Scholar Rock: Consultancy, Equity Ownership; Centocor/Janssen: Patents &amp; Royalties: abxicimab; Accumetrics/Instrumentation Laboratory: Patents &amp; Royalties: VerifyNow assay.

Thrombotic biomarkers for risk prediction of malignant disease recurrence in patients with early stage breast cancer.

In cancer patients, hypercoagulability is a common finding. It has been associated with an increased risk of venous thromboembolism, but also to tumor proliferation and progression. In this prospective study of a large cohort of breast cancer patients, we aimed to evaluate whether pre-chemotherapy abnormalities in hemostatic biomarkers levels: (i) are associated with breast cancer-specific clinico-pathological features; and (ii) can predict for disease recurrence. D-dimer, fibrinogen, prothrombin fragment 1+2, and FVIIa/antithrombin levels were measured in 701 early-stage resected breast cancer patients candidate to adjuvant chemotherapy and prospectively enrolled in the HYPERCAN study. Significant prognostic parameters for disease recurrence were identified by Cox regression multivariate analysis and used for generating a risk assessment model. Pre-chemotherapy D-dimer, fibrinogen, and pro-thrombin fragment 1+2 levels were significantly associated with tumor size and lymph node metastasis. After 3.4 years of follow up, 71 patients experienced a recurrence. Cox multivariate analysis identified prothrombin fragment 1+2, tumor size, and Luminal B HER2-negative or triple negative molecular subtypes as independent risk factors for disease recurrence. Based on these variables, we generated a risk assessment model that significantly differentiated patients at low- and high-risk of recurrence (cumulative incidence: 6.2 vs. 20.7%; Hazard Ratio=3.5; P<0.001). Our prospective clinical and laboratory data from the HYPERCAN study were crucial for generating a scoring model for assessing risk of disease recurrence in resected breast cancer patients, candidate to systemic chemotherapy. This finding stimulates future investigations addressing the role of plasma prothrombin fragment 1+2 in the management of breast cancer patients to provide the rationale for new therapeutic strategies. (The HYPERCAN study is registered at clinicaltrials.gov identifier 02622815.)

Fibrinogen-Based Hydrogel Modulus and Ligand Density Effects on Cell Morphogenesis in Two-Dimensional and Three-Dimensional Cell Cultures.

There is a need to further explore the convergence of mechanobiology and dimensionality with systematic investigations of cellular response to matrix mechanics in 2D and 3D cultures. Here, a semisynthetic hydrogel capable of supporting both 2D and 3D cell culture is applied to investigate cell response to matrix modulus and ligand density. The culture materials are fabricated from adducts of polyethylene glycol (PEG) or PluronicF127 and fibrinogen fragments, formed into hydrogels by free-radical polymerization, and characterized by shear rheology. Control over the modulus of the materials is accomplished by changing the concentration of synthetic PEG-diacrylate crosslinker (0.5% w/v), and by altering the molecular length of the PEG (10 and 20 kDa). Control over ligand density is accomplished by changing fibrinogen concentrations from 3 to 12 mg mL-1 . In 2D culture, cell motility parameters, including cell speed and persistence time are significantly increased with increasing modulus. In both 2D and 3D culture, cells express vinculin and there is evidence of focal adhesion formation in the high stiffness materials. The modulus- and ligand-dependent morphogenesis response from the cells in 2D culture is contradictory to the same measured response in 3D culture. In 2D culture, anchorage-dependent cells become more elongated and significantly increase their size with increasing ligand density and matrix modulus. In 3D culture, the same anchorage-dependent cells become less spindled and significantly reduce their size in response to increasing ligand density and matrix modulus. These differences arise from dimensionality constraints, most notably the encapsulation of cells in a non-porous hydrogel matrix. These insights underscore the importance of mechanical properties in regulating cell morphogenesis in a 3D culture milieu. The versatility of the hydrogel culture environment further highlights the significance of a modular approach when developing materials that aim to optimize the cell culture environment.

The Interaction of Cross-Linked Fibrin Fragment D-Dimer with αIIbβ3 Requires Receptor Activation, Occurs at Sites Other Than the γ-404-411 Sequence, and Contributes to Clot Retraction

Background: The interaction between the fibrinogen (fbg) γ-chain sequence 404-411 and the ‘RGD pocket’ formed by αIIb and β3 plays a major role in platelet (P) aggregation. It requires activation of αIIbβ3 when fbg is in solution, but not when fbg is immobilized. Less is known about the interaction of Ps with cross-linked fibrin, which presumably is the dominant form of fibrin(ogen) in human thrombi and the form that participates in clot retraction. αIIbβ3 is required for clot retraction since it is decreased or absent in Glanzmann thrombasthenia patients who lack a functional receptor. Paradoxically, clot retraction is essentially normal with fbg lacking γ-408-411 or in the presence EDTA, which eliminates fbg binding to the RGD pocket. We recently studied the interaction of αIIbβ3 with fbg fragments D100 (which has γ-404-411) and 'D98' (which essentially lacks γ-406-411) to identify fbg binding sites on αIIbβ3 other than the RGD pocket. We found that: 1. activated, but not unactivated, Ps adhere well to immobilized 'D98;' 2. cells expressing normal αIIbβ3 could bind to: a) fbg in the absence of EDTA, but not in the presence of EDTA, b) D100 in the absence or presence of EDTA, c) 'D98' in the presence, but not the absence, of EDTA; 3. cells expressing constitutively active αIIbβ3 mutants, but not cells expressing normal αIIbβ3, adhere well to immobilized 'D98.' These data support there being two separate mechanisms of binding, one involving the interaction of γ-404-411 (in fbg and D100) with the αIIbβ3 RGD binding pocket, and another involving a site on D100 and 'D98' (but cryptic in fbg) that interacts with a site on αIIbβ3 exposed by receptor activation or induced by EDTA.We have now studied adhesion of αIIbβ3 to the cross-linked fibrin fragment D-dimer (D-d). We predicted it to be similar to the interaction of ‘D98‘ with αIIbβ3 because γ-K406, which is important in the binding of fbg to αIIb, participates in the antiparallel reciprocal factor XIIIa-mediated transglutaminase crosslinks between K406 and Q398 on two different γ-chains, and so is unlikely to be able to interact with the RGD pocket.Methods: D-d, purified from cross-linked fibrin after plasmin digestion, was obtained from Haematologic Technologies and found to contain a dominant band with the expected Mr and immunoblot pattern using antibodies to the individual fbg chains. Fibrinogen, 'D98,' and HEK293 cells expressing normal and mutated αIIbβ3 were prepared as we previously described (Zafar et al., Blood Advances 2017), and the adhesion assays were conducted as described in that paper. Clot retraction was performed by adding BSA, 'D98,' or D-d (200 µg/ml) to washed Ps (2 X 108/ml) + thrombin (0.2 U/ml) in a glass aggregometer cuvette.Results: αIIbβ3-mediated adhesion to immobilized D-d (10 µg/ml coating concentration) was similar to adhesion to 'D98' in that 1. Unactivated P adhered well to fbg, but poorly to 'D98' and D-d (n=4); 2. Activated Ps (10 µM thrombin receptor activating peptide) adhere ~2-fold better than unactivated Ps to fbg, but ~10-fold better to 'D98' and D-d (p<0.001 for unactivated vs activated Ps with both 'D98' and D-d; n=4); 3. HEK293 cells expressing normal αIIbβ3 (αIIbβ3-HEK) adhere well to fibrinogen, but not 'D98' and D-d; 4. HEK293 cells expressing the constitutively active aIIbF992A/F993Aβ3 receptor [αIIb(FF)β3-HEK] bound well to fbg, 'D98, and D-d (p<0.001 for both 'D98' and D-d compared to normal αIIbβ3; n=5); 5. EDTA treatment of Ps results in loss of adhesion to fbg, but significantly enhanced adhesion to 'D98' and D-d (p<0.05 for each ligand with and without EDTA; n=4); 6. EDTA treatment of αIIbβ3-HEK results in loss of adhesion to fbg, but significantly enhanced adhesion to both 'D98' and D-d (p<0.01 for each ligand with and without EDTA; n=5); 7. Adhesion of αIIb(FF)β3-HEK to D-d is like adhesion to 'D98' in being inhibitable by mAbs 7E3 and 10E5, eptifibatide, tirofiban, and RUC-4 (p<0.01 for all); n=4); 8. Adhesion of αIIb(FF)β3-HEK to D-d is similar to adhesion to 'D98' in being inhibitable by mutating β3 D119 to A in the MIDAS (p<0.01; n=6); 9. EDTA-induced adhesion of HEK-αIIbβ3 to both 'D98' and D-d is not inhibitable by mutating β3 D119 to A. 10. 'D98' and D-d inhibited clot retraction, but BSA did not.Conclusion: We conclude that cross-linked fibrin fragment D-dimer, like fibrinogen fragment ‘D98‘, interacts with activated αIIbβ3 via sites other than the γ-404-411 peptide, and that this interaction contributes to clot retraction. DisclosuresColler:Rockefeller University: Patents & Royalties: RUC-4; Platelet Biogenesis: Consultancy; Centocor: Patents & Royalties: Abciximab; CeleCor: Consultancy, Equity Ownership; Accumetrics: Patents & Royalties: VerifyNow Assays; Scholar Rock: Consultancy, Equity Ownership.

Activation of coagulation and endothelium with concurrent impairment of anticoagulant mechanisms in patients with typhoid fever

Typhoid fever caused by Salmonella Typhi remains a major burden worldwide. Gastrointestinal bleeding can be seen in up to 10 percent of patients and may be fatal. The coagulopathy, which may be the driver of this severe complication in patients with typhoid fever, however is ill defined. The aim of this study was to evaluate the activation of coagulation, anticoagulation, and fibrinolysis in patients with acute typhoid fever. Parameters of coagulation and fibrinolysis were measured in 28 hospitalized patients with culture-confirmed or PCR-confirmed typhoid fever and compared to 38 age- and sex-matched healthy volunteers. Patients demonstrated activation of the coagulation system, as reflected by elevated in vitro thrombin generation and high plasma levels of fibrinogen, D-dimer and prothrombin fragment F1 + 2 in concert with consumption of coagulation factors resulting in a prolonged prothrombin-time and activated-partial-thromboplastin-time. Concurrently, the anticoagulant proteins, protein C and antithrombin, were significantly lower in comparison to healthy controls. Patients also demonstrated evidence of activation and inhibition of fibrinolysis and a marked activation of endothelial cells. The extent of coagulation activation was associated with the course of the disease, repeated testing during convalescence showed a return toward normal values. Activation of coagulation is an important clinical feature of typhoid fever and is associated with severity of disease.

Urinary CE-MS peptide marker pattern for detection of solid tumors

Urinary profiling datasets, previously acquired by capillary electrophoresis coupled to mass-spectrometry were investigated to identify a general urinary marker pattern for detection of solid tumors by targeting common systemic events associated with tumor-related inflammation. A total of 2,055 urinary profiles were analyzed, derived from a) a cancer group of patients (n = 969) with bladder, prostate, and pancreatic cancers, renal cell carcinoma, and cholangiocarcinoma and b) a control group of patients with benign diseases (n = 556), inflammatory diseases (n = 199) and healthy individuals (n = 331). Statistical analysis was conducted in a discovery set of 676 cancer cases and 744 controls. 193 peptides differing at statistically significant levels between cases and controls were selected and combined to a multi-dimensional marker pattern using support vector machine algorithms. Independent validation in a set of 635 patients (293 cancer cases and 342 controls) showed an AUC of 0.82. Inclusion of age as independent variable, significantly increased the AUC value to 0.85. Among the identified peptides were mucins, fibrinogen and collagen fragments. Further studies are planned to assess the pattern value to monitor patients for tumor recurrence. In this proof-of-concept study, a general tumor marker pattern was developed to detect cancer based on shared biomarkers, likely indicative of cancer-related features.

Putative salivary protein biomarkers for the diagnosis of oral lichen planus: a case-control study

BackgroundSalivary protein biomarkers for screening and diagnosis of oral lichen planus (OLP) are not well-defined. The objective of this study was to identify putative protein biomarkers for OLP using proteomic approaches.MethodsPooled unstimulated whole saliva was collected from five OLP patients and five healthy control participants. Saliva samples were then subjected to two-dimensional gel electrophoresis, followed by mass spectrometry to identify putative protein biomarkers. Subsequently, a subset of these putative biomarkers were validated in 24 OLP patients and 24 age-matched healthy control subjects, using an enzyme-linked immunosorbent assay (ELISA). Immunoblotting analyses were then performed in 3 pairs of age- and sex-matched OLP patients and healthy controls to confirm results from the ELISA study.ResultsThirty-one protein spots were identified, corresponding to 20 unique proteins. Notably, fibrinogen fragment D and complement component C3c exhibited increased expression in OLP patients, while cystatin SA exhibited decreased expression in OLP patients, compared with healthy control subjects. ELISA analyses indicated increased expression of fibrinogen fragment D and complement component C3c, and decreased expression of cystatin SA, in the saliva of OLP patients. Statistical differences in the expression of salivary complement C3c were observed between OLP patients and healthy control subjects. Immunoblotting analyses confirmed the results of our ELISA study.ConclusionComplement C3c, fibrinogen fragment D and cystatin SA may serve as salivary biomarkers for screening and/or diagnosis of OLP.

Diminished coagulation capacity assessed by calibrated automated thrombography during acute Puumala hantavirus infection.

: Coagulation abnormalities are associated with Puumala-virus-induced hemorrhagic fever with renal syndrome (PUUV-HFRS). We evaluated the coagulation capacity of plasma during acute PUUV-HFRS by measuring thrombin generation using calibrated automated thrombography (CAT). The study cohort comprised 27 prospectively collected, consecutive, hospital-treated patients with acute PUUV infection. Blood samples were drawn in the acute phase and at the control visit approximately 5 weeks later. To evaluate thrombin generation, the lag time of initiation, endogenous thrombin potential (ETP), and peak and time to peak thrombin concentration were assessed by CAT in platelet poor plasma without corn trypsin inhibitor. Plasma levels of D-dimer, fibrinogen and prothrombin fragments (F1 + 2) were also evaluated. When the acute phase was compared with the control phase, ETP was decreased (median 1154 nmol/l/min, range 67-1785 vs. median 1385 nmol/l/min, range 670-1970; P < 0.001), while the lag time was prolonged (median 3.8 min, range 2.1-7.7 vs. median 2.9 min, range 2.0-4.1; P < 0.001). Low ETP correlated with low peak thrombin concentration (r = 0.833, P < 0.001). Prolonged time to peak associated with the lag time (r = 0.78, P < 0.001). ETP was associated with thrombocytopenia (r = 0.472, P = 0.015) and weakly with fibrinogen level (r = 0.386, P = 0.047). The measured CAT parameters did not associate with D-dimer and F1 + 2 levels. Decreased ETP together with low peak and prolonged lag time indicate decreased plasma potential for thrombin generation in vitro. Together with low platelet count and enhanced fibrinolysis, this further refers to altered blood coagulation and increased propensity toward bleeding in acute PUUV-HFRS.