Roles of fibrin α- and γ-chain specific cross-linking by FXIIIa in fibrin structure and function

Factor XIII is responsible for the cross-linking of fibrin γ-chains in the early stages of clot formation, whilst α-chain cross-linking occurs at a slower rate. Although γ- and α-chain cross-linking was previously shown to contribute to clot stiffness, the role of cross-linking of both chains in determining clot structure is currently unknown. Therefore, the aim of this study was to determine the role of individual α- and γ-chain cross-linking during clot formation, and its effects on clot structure. We made use of a recombinant fibrinogen (γQ398N/Q399N/K406R), which does not allow for γ-chain cross-linking. In the absence of cross-linking, intact D-D interface was shown to play a potential role in fibre appearance time, clot stiffness and elasticity. Cross-linking of the fibrin α-chain played a role in the thickening of the fibrin fibres over time, and decreased lysis rate in the absence of α2-antiplasmin. We also showed that α-chain cross-linking played a role in the timing of fibre appearance, straightening fibres, increasing clot stiffness and reducing clot deformation. Cross-linking of the γ-chain played a role in fibrin fibre appearance time and fibre density. Our results show that α- and γ-chain cross-linking play independent and specific roles in fibrin clot formation and structure

A novel method to quantify fibrin-fibrin and fibrin-α2AP cross-links in thrombi formed from human trauma patient plasma.

The widespread use of the anti-fibrinolytic agent, tranexamic acid (TXA), interferes with the quantification of fibrinolysis by dynamic laboratory assays such as clot lysis, making it difficult to measure fibrinolysis in many trauma patients. At the final stage of coagulation, Factor XIIIa (FXIIIa) catalyses the formation of fibrin-fibrin and fibrin-α2-antiplasmin (α2AP) cross-links which increases clot mechanical strength and resistance to fibrinolysis. Here, we develop a method to quantify fibrin-fibrin and fibrin-α2AP cross-links that avoids the challenges posed by TXA in determining fibrinolytic resistance in conventional assays. Fibrinogen alpha chain (FGA-FGA), fibrinogen gamma chain (FGG-FGG) and FGA-α2AP cross-links were quantified using liquid-chromatography-mass spectrometry (LC-MS) and parallel reaction monitoring (PRM) in paired plasma samples from trauma patients pre- and post-fibrinogen replacement. Differences in the abundance of cross-links in trauma patients receiving cryoprecipitate (cryo) or fibrinogen concentrate (Fg-C) were analysed. The study found that the abundance of cross-links was significantly increased in trauma patients post-cryo, but not Fg-C, transfusion (p < 0.0001). The abundance of cross-links was positively correlated with the toughness of individual fibrin fibres, the peak thrombin concentration and FXIII antigen (p < 0.05). We have developed a novel method that allows us to quantify fibrin cross-links in trauma patients who have received TXA, providing an indirect measure of fibrinolytic resistance. Using this novel approach we have avoided the effect of TXA and shown that cryo increases fibrin-fibrin and fibrin-α2AP cross-linking when compared to Fg-C, highlighting the importance of FXIII in clot formation and stability in trauma patients

Immobilized fibrinogen activates human platelets through GPVI

GPVI, a major platelet activation receptor for collagen and fibrin, is considered as a particularly promising safe antithrombotic target. In this study, we show that human GPVI signals upon platelet adhesion to fibrinogen. Full spreading of human platelets on fibrinogen is abolished in platelets from GPVI-deficient patients suggesting that fibrinogen activates platelets through GPVI. While mouse platelets fail to spread on fibrinogen, human-GPVI-transgenic mouse platelets show full spreading and increased Ca2+ signalling through the tyrosine kinase Syk. Direct binding of fibrinogen to human GPVI was shown by surface plasmon resonance and by increased adhesion of human GPVI-transfected Rbl-2H3 cells to fibrinogen relative to mock-transfected cells. Blockade of human GPVI with the Fab of the monoclonal antibody 9O12 impairs platelet aggregation on preformed platelet aggregates in flowing blood independent of collagen and fibrin exposure. These results demonstrate that human GPVI binds to immobilized fibrinogen and show that this contributes to platelet spreading and platelet aggregation under flow

Pharmacodynamic Modeling of Anti-Cancer Activity of Tetraiodothyroacetic Acid in a Perfused Cell Culture System

Unmodified or as a poly[lactide-co-glycolide] nanoparticle, tetraiodothyroacetic acid (tetrac) acts at the integrin αvβ3 receptor on human cancer cells to inhibit tumor cell proliferation and xenograft growth. To study in vitro the pharmacodynamics of tetrac formulations in the absence of and in conjunction with other chemotherapeutic agents, we developed a perfusion bellows cell culture system. Cells were grown on polymer flakes and exposed to various concentrations of tetrac, nano-tetrac, resveratrol, cetuximab, or a combination for up to 18 days. Cells were harvested and counted every one or two days. Both NONMEM VI and the exact Monte Carlo parametric expectation maximization algorithm in S-ADAPT were utilized for mathematical modeling. Unmodified tetrac inhibited the proliferation of cancer cells and did so with differing potency in different cell lines. The developed mechanism-based model included two effects of tetrac on different parts of the cell cycle which could be distinguished. For human breast cancer cells, modeling suggested a higher sensitivity (lower IC50) to the effect on success rate of replication than the effect on rate of growth, whereas the capacity (Imax) was larger for the effect on growth rate. Nanoparticulate tetrac (nano-tetrac), which does not enter into cells, had a higher potency and a larger anti-proliferative effect than unmodified tetrac. Fluorescence-activated cell sorting analysis of harvested cells revealed tetrac and nano-tetrac induced concentration-dependent apoptosis that was correlated with expression of pro-apoptotic proteins, such as p53, p21, PIG3 and BAD for nano-tetrac, while unmodified tetrac showed a different profile. Approximately additive anti-proliferative effects were found for the combinations of tetrac and resveratrol, tetrac and cetuximab (Erbitux), and nano-tetrac and cetuximab. Our in vitro perfusion cancer cell system together with mathematical modeling successfully described the anti-proliferative effects over time of tetrac and nano-tetrac and may be useful for dose-finding and studying the pharmacodynamics of other chemotherapeutic agents or their combinations

Religious Liberty in a Pluralistic Society (2nd ed.)

The second edition of Religious Liberty in a Pluralistic Society offers the same structure and thorough coverage of the law of religious liberty as the first edition, along with a new conceptual framework for approaching the religious liberty jurisprudence of the Supreme Court. The first four chapters offer a history of law and religion in the United States that extends from the framing of the Constitution to the early 1920s. Chapters Six through Thirteen examine the statute and case law governing religious liberty in a variety of settings and areas of law, including education, the workplace, tax, the courtroom, property, and the corporate boardroom. The few pronouncements of the United States Supreme Court in each of these areas serve as the anchors for thorough examination of the law of religious liberty in the state and lower federal courts. Ariens and Destro have reorganized Chapter Five, which examines the Supreme Court\u27s efforts to craft a constitutional law of religious liberty since the 1940s. The new conceptual framework is based on the language and structure of the First Amendment, and is designed to help the reader understand and apply the rules the Court has developed in this important area of constitutional law. New in the notes to Chapter Five are references to comparative and international materials. The materials are updated through 2001, and a number of cases are more tightly edited than in the first edition. A revised teacher\u27s manual with sample course outlines and problems will be available.https://scholarship.law.edu/fac_books/1036/thumbnail.jp

Religious Liberty in a Pluralistic Society (2nd ed.)

The second edition of Religious Liberty in a Pluralistic Society offers the same structure and thorough coverage of the law of religious liberty as the first edition, along with a new conceptual framework for approaching the religious liberty jurisprudence of the Supreme Court. The first four chapters offer a history of law and religion in the United States that extends from the framing of the Constitution to the early 1920s. Chapters Six through Thirteen examine the statute and case law governing religious liberty in a variety of settings and areas of law, including education, the workplace, tax, the courtroom, property, and the corporate boardroom. The few pronouncements of the United States Supreme Court in each of these areas serve as the anchors for thorough examination of the law of religious liberty in the state and lower federal courts. Ariens and Destro have reorganized Chapter Five, which examines the Supreme Court\u27s efforts to craft a constitutional law of religious liberty since the 1940s. The new conceptual framework is based on the language and structure of the First Amendment, and is designed to help the reader understand and apply the rules the Court has developed in this important area of constitutional law. New in the notes to Chapter Five are references to comparative and international materials. The materials are updated through 2001, and a number of cases are more tightly edited than in the first edition. A revised teacher\u27s manual with sample course outlines and problems will be available.https://scholarship.law.edu/fac_books/1036/thumbnail.jp