Polyphosphate (polyP) binds to fibrin(ogen) and alters fibrin structure, generating a heterogeneous network composed of 'knots' interspersed by large pores. Here we show platelet-derived polyP elicits similar structural changes in fibrin and examine the mechanism by which polyP alters fibrin structure. Polymerisation of fibrinogen with thrombin and CaCl2 was studied using spinning disk confocal (SDC) microscopy. PolyP delayed fibrin polymerisation generating shorter protofibrils emanating from a nucleus-type structure. Consistent with this, cascade blue-polyP accumulated in fibrin 'knots'. Protofibril formation was visualized by atomic force microscopy (AFM) ± polyP. In the presence of polyP abundant monomers of longer length were visualised by AFM, suggesting that polyP binds to monomeric fibrin. Shorter oligomers form in the presence of polyP, consistent with the stunted protofibrils visualised by SDC microscopy. We examined whether these structural changes induced by polyP alter fibrin's viscoelastic properties by rheometry. PolyP reduced the stiffness (G') and ability of the fibrin network to deform plastically G'', but to different extents. Consequently, the relative plastic component (loss tangent (G''/G')) was 61 % higher implying that networks containing polyP are less stiff and more plastic. Local rheological measurements, performed using magnetic tweezers, indicate that the fibrin dense knots are stiffer and more plastic, reflecting the heterogeneity of the network. Our data show that polyP impedes fibrin polymerisation, stunting protofibril growth producing 'knotted' regions, which are rich in fibrin and polyP. Consequently, the mechanical properties of the fibrin network are altered resulting in clots with overall reduced stiffness and increased ability to deform plastically.

Platelet concentrates are extensively utilized in the medical and dental field to promote tissue regeneration. The profusion of endogenous growth factors in platelets α-granules transmit their use for enhanced wound healing. However, little attention has been given to study their antimicrobial potential. This study was conducted to assess the antibacterial and antifungal property of platelet-rich fibrin (PRF) and PRF matrix (PRFM).

The diagnosis of early and aggressive types of cancer is important for providing effective cancer therapy. Cancer-induced fibrin clots exist only within lesions. Previously, we developed a monoclonal antibody (clone 102-10) that recognizes insoluble fibrin but not fibrinogen or soluble fibrin and confirmed that fibrin clots form continuously in various cancers. Here, we describe the development of a Fab fragment probe of clone 102-10 for tumour imaging. The distribution of 102-10 Fab was investigated in genetically engineered mice bearing pancreatic ductal adenocarcinoma (PDAC), and its effect on blood coagulation was examined. Immunohistochemical and ex vivo imaging revealed that 102-10 Fab was distributed selectively in fibrin clots in PDAC tumours 3 h after injection and that it disappeared from the body after 24 h. 102-10 Fab had no influence on blood coagulation or fibrinolysis. Tumour imaging using anti-fibrin Fab may provide a safe and effective method for the diagnosis of invasive cancers by detecting fibrin clots in tumour stroma.

The space-filling fibrin network is a major part of clots and thrombi formed in blood. Fibrin polymerization starts when fibrinogen, a plasma protein, is proteolytically converted to fibrin, which self-assembles to form double-stranded protofibrils. When reaching a critical length, these intermediate species aggregate laterally to transform into fibers arranged into branched fibrin network. We combined multiscale modeling in silico with atomic force microscopy (AFM) imaging to reconstruct complete atomic models of double-stranded fibrin protofibrils with γ-γ crosslinking, A:a and B:b knob-hole bonds, and αC regions-all important structural determinants not resolved crystallographically. Structures of fibrin oligomers and protofibrils containing up to 19 monomers were successfully validated by quantitative comparison with high-resolution AFM images. We characterized the protofibril twisting, bending, kinking, and reversibility of A:a knob-hole bonds, and calculated hydrodynamic parameters of fibrin oligomers. Atomic structures of protofibrils provide a basis to understand mechanisms of early stages of fibrin polymerization.

Cancer-induced blood coagulation in human tumour generates insoluble fibrin (IF)-rich cancer stroma in which uneven monoclonal antibody (mAb) distribution reduce the potential effectiveness of mAb-mediated treatments. Previously, we developed a mAb that reacts only with IF and not with fibrinogen (FNG) or the fibrin degradation product (FDP). Although IF, FNG and FDP share same amino acid sequences, the mAb is hardly neutralised by FNG and FDP in circulation and accumulates in fibrin clots within tumour tissue. Here, we created an antibody drug conjugate (ADC) using the anti-IF mAb conjugated with a chemotherapy payload (IF-ADC). The conjugate contains a linker severed specifically by plasmin (PLM), which is activated only on binding to IF. Imaging mass spectrometry showed the substantial intratumour distribution of the payload following the IF-ADC injection into mice bearing IF-rich 5-11 xenografts derived from pancreatic tumours of LSL-KrasG12D/+; LSL-Trp53R172H/+; Ptf1a-Cre (KPC) mice. IF-ADC treatment significantly extended the survival of the KPC mice. These data suggest that conjugating chemotherapy drugs to this IF-specific mAb could represent an effective means of treating stroma-rich tumours.

The purpose of this study was to investigate the effects of platelet-rich fibrin (PRF) modified with bioactive radiopacifiers-nanohydroxyapatite (nHA) and dentin chips (DC) on odontoblastic differentiation in human dental pulp cells (HDPCs).

Fibrin, a product of the blood coagulation cascade, accompanies many type 1 immune responses, including delayed-type hypersensitivity, autoimmunity, and graft rejection. In those settings, fibrin is thought to exacerbate inflammation and disease. Here, we evaluate roles for coagulation during infection with Toxoplasma gondii, a pathogen whose control requires robust type 1 immunity. We establish that fibrin prevents infection-stimulated blood loss, thereby performing a protective function that is essential for survival. Remarkably, fibrin does not simply protect against vascular damage caused directly by the infectious agent, but rather, protects against hemorrhage evoked by interferon-gamma, a critical mediator of type 1 immunity. This finding, to our knowledge, is the first to document a beneficial role for coagulation during type 1 immunity, and suggests that fibrin deposition protects host tissue from collateral damage caused by the immune system as it combats infection.

Platelet-rich fibrin (PRF) membranes can preserve alveolar ridge dimension after tooth extraction. Thus, it can be presumed that PRF suppresses the catabolic events that are caused by osteoclastic bone resorption.

The autologous platelet concentrates (PCs), such as platelet-rich fibrin (PRF) and platelet-rich fibrin matrix (PRFM), are processed through different centrifugation protocols, which can affect their biological properties and in turn influence treatment outcome. The pH value can influence the process of wound healing directly or indirectly. Hence, a study was conducted to evaluate fibrin network pattern, initial pH of obtained matrix, and its changes during degradation matrix and to determine platelet and leukocyte count in PRF and PRFM.

Several proteins are known to bind to a fibrin network and to change clot properties or function. In this study we aimed to get an overview of fibrin clot-bound plasma proteins. A plasma clot was formed by adding thrombin, CaCl(2) and aprotinin to citrated platelet-poor plasma and unbound proteins were washed away with Tris-buffered saline. Non-covalently bound proteins were extracted, separated with 2D gel electrophoresis and visualized with Sypro Ruby. Excised protein spots were analyzed with mass spectrometry. The identity of the proteins was verified by checking the mass of the protein, and, if necessary, by Western blot analysis. Next to established fibrin-binding proteins we identified several novel fibrin clot-bound plasma proteins, including α(2)-macroglobulin, carboxypeptidase N, α(1)-antitrypsin, haptoglobin, serum amyloid P, and the apolipoproteins A-I, E, J, and A-IV. The latter six proteins are associated with high-density lipoprotein particles. In addition we showed that high-density lipoprotein associated proteins were also present in fibrinogen preparations purified from plasma. Most plasma proteins in a fibrin clot can be classified into three groups according to either blood coagulation, protease inhibition or high-density lipoprotein metabolism. The presence of high-density lipoprotein in clots might point to a role in hemostasis.

Fibrin hydrogels made by self-assembly of fibrinogen obtained from human plasma have shown excellent biocompatible and biodegradable properties and are widely used in regenerative medicine. The fibrinogen self-assembly process can be triggered under physiological conditions by the action of thrombin, allowing the injection of pregel mixtures that have been used as cell carriers, wound-healing systems, and bio-adhesives. However, access to fibrinogen from human plasma is expensive and fibrin gels have limited mechanical properties, which make them unsuitable for certain applications. One solution to these problems is to obtain composite gels made of fibrin and other polymeric compounds that improve their mechanical properties and usage. Herein, we prepared composite hydrogels made by the self-assembly of fibrinogen together with Fmoc-FF (Fmoc-diphenylalanine) and Fmoc-RGD (Fmoc-arginine-glycine-aspartic acid). We have shown that the mixture of these three peptides co-assembles and gives rise to a unique type of supramolecular fiber, whose morphology and mechanical properties can be modulated. We have carried out a complete characterization of these materials from chemical, physical, and biological points of view. Composite gels have improved mechanical properties compared to pure fibrin gels, as well as showing excellent biocompatibility ex vivo. In vivo experiments have shown that these gels do not cause any type of inflammatory response or tissue damage and are completely resorbed in short time, which would enable their use as vehicles for cell, drug, or growth factor release.

The study included 60 patients according to sample size calculation, recruited from patients seeking tooth extraction at oral and maxillofacial surgery clinic at Umm Al-Qura University, Faculty of Dentistry. Patients were divided into three groups. Group Ӏ included 20 patients managed by advanced platelet-rich fibrin after extraction. Group ӀӀ included 20 patients managed by leukocyte-platelet-rich fibrin after tooth extraction. Group ӀII included 20 patients left without any addition. Each group was further subdivided into surgical and nonsurgical extraction. Afterwards, patients in each group were assessed for postextraction pain by VAS, number of analgesics, and early soft tissue healing by LWHI.

To determine the efficacy of usage of topical autologous platelet rich fibrin (PRF) in improving outcomes of myringoplasty regarding graft uptake and hearing improvement.

In knee arthroscopic surgery, fibrin clot (FC) and leukocyte-rich platelet-rich fibrin (L-PRF) may be used in augmentation for meniscal repair. Studies have investigated growth factors released from FC and L-PRF; however, it is difficult to compare FC and L-PRF between different studies. Direct comparison of growth factors that may support meniscal healing released from FC and L-PRF may be beneficial in deciding whether to use FC or L-PRF. If no significant difference is seen, the surgeon may decide to use FC which is easier to prepare compared to L-PRF. The purpose of this pilot study is to investigate the release amount and pattern of basic fibroblast growth factor (bFGF), platelet-derived growth factor AB (PDGF-AB), transforming growth factor β1 (TGF-β1), vascular endothelial growth factor (VEGF), and stromal cell-derived factor 1 (SDF-1) from FC and L-PRF.

Altered fibrin fiber structure is linked to pathologic states, including coronary heart disease, ischemic stroke, and atherosclerosis. However, several different techniques are commonly utilized for studying fibrin structures, and comparison of results obtained using different techniques can be challenging due to lack of standardization.

Fibrin hydrogel is a central biological material in tissue engineering and drug delivery applications. As such, fibrin is typically combined with cells and biomolecules targeted to the regenerated tissue. Previous studies have analyzed the release of different molecules from fibrin hydrogels; however, the effect of embedded cells on the release profile has yet to be quantitatively explored. This study focused on the release of Fluorescein isothiocyanate (FITC)-dextran (FD) 250 kDa from fibrin hydrogels, populated with different concentrations of fibroblast or endothelial cells, during a 48-h observation period. The addition of cells to fibrin gels decreased the overall release by a small percentage (by 7-15% for fibroblasts and 6-8% for endothelial cells) relative to acellular gels. The release profile was shown to be modulated by various cellular activities, including gel degradation and physical obstruction to diffusion. Cell-generated forces and matrix deformation (i.e., densification and fiber alignment) were not found to significantly influence the release profiles. This knowledge is expected to improve fibrin integration in tissue engineering and drug delivery applications by enabling predictions and ways to modulate the release profiles of various biomolecules.

Circulating levels of Brain Derived Neurotrophic Factor (BDNF) are lower in coronary heart disease (CHD) than in healthy subjects and are associated with coronary events and mortality. However, the mechanism(s) underling this association is not fully understood. We hypothesize that BDNF may influence fibrin fiber structure and clot stability, favoring clot lysis and thrombus resolution. We showed that recombinant BDNF (rh-BDNF) influenced with clot formation in a concentration-dependent manner in both purified fibrinogen and plasma from healthy subjects. In particular, rh-BDNF reduced the density of fibrin fibers, the maximum clot firmness (MCF) and the maximum clot turbidity, and affected the lysis of clot. In addition, both thrombin and reptilase clotting time were prolonged by rh-BDNF, despite the amount of thrombin formed was greater. Intriguingly, CHD patients had lower levels of BDNF, greater fibrin fibers density, higher MCF than control subjects, and a negative correlation between BDNF and MCF was found. Of note, rh-BDNF markedly modified fibrin clot profile restoring physiological clot morphology in CHD plasma. In conclusion, we provide evidence that low levels of BDNF correlate with the formation of bigger thrombi (in vitro) and that this effect is mediated, at least partially, by the alteration of fibrin fibers formation.

The differences between coarse and fine fibrin clots first reported by Ferry have been interpreted in terms of nonspecific ionic strength effects for nearly 50 years and have fostered the notion that fibrin polymerization is largely controlled by electrostatic forces. Here we report spectroscopic and electron microscopy studies carried out in the presence of different salts that demonstrate that this long-held interpretation needs to be modified. In fact, the differences are due entirely to the specific binding of Cl- to fibrin fibers and not to generic ionic strength or electrostatic effects. Binding of Cl- opposes the lateral aggregation of protofibrils and results in thinner fibers that are also more curved than those grown in the presence of inert anions such as F-. The effect of Cl- is pH dependent and increases at pH > 8.0, whereas fibers grown in the presence of F- remain thick over the entire pH range from 6.5 to 9.0. From the pH dependence of the Cl- effect it is suggested that the anion exerts its role by increasing the pKa of a basic group ionizing around pH 9.2. The important role of Cl- in structuring the fibrin clot also clarifies the role played by the release of fibrinopeptide B, which leads to slightly thicker fibers in the presence of Cl- but actually reduces the size of the fibers in the presence of F-. This effect becomes more evident at high, close to physiological concentrations of fibrinogen. We conclude that Cl- is a basic physiological modulator of fibrin polymerization and acts to prevent the growth of thicker, stiffer, and straighter fibers by increasing the pKa of a basic group. This discovery opens new possibilities for the design of molecules that can specifically modify the clot structure by targeting the structural domains responsible for Cl- binding to fibrin.

Activation of innate immunity and deposition of blood-derived fibrin in the central nervous system (CNS) occur in autoimmune and neurodegenerative diseases, including multiple sclerosis (MS) and Alzheimer's disease (AD). However, the mechanisms that link disruption of the blood-brain barrier (BBB) to neurodegeneration are poorly understood, and exploration of fibrin as a therapeutic target has been limited by its beneficial clotting functions. Here we report the generation of monoclonal antibody 5B8, targeted against the cryptic fibrin epitope γ377-395, to selectively inhibit fibrin-induced inflammation and oxidative stress without interfering with clotting. 5B8 suppressed fibrin-induced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation and the expression of proinflammatory genes. In animal models of MS and AD, 5B8 entered the CNS and bound to parenchymal fibrin, and its therapeutic administration reduced the activation of innate immunity and neurodegeneration. Thus, fibrin-targeting immunotherapy inhibited autoimmunity- and amyloid-driven neurotoxicity and might have clinical benefit without globally suppressing innate immunity or interfering with coagulation in diverse neurological diseases.

Platelet products play a fundamental role in the process of healing. The new generation of platelet-rich fibrin (PRF), namely advanced PRF (A-PRF), has different biological and mechanical properties compared to those of leukocyte-PRF (L-PRF). This study aimed to compare the effects of L-PRF and A-PRF on the viability and migration of human gingival fibroblasts (HGFs).