Nanomechanics of Blood Clot and Thrombus Formation

Mechanical properties have been extensively studied in pure elastic or viscous materials; however, most biomaterials possess both physical properties in a viscoelastic component. How the biomechanics of a fibrin clot is related to its composition and the microenvironment where it is formed is not yet fully understood. This review gives an outline of the building mechanisms for blood clot mechanical properties and how they relate to clot function. The formation of a blood clot in health conditions or the formation of a dangerous thrombus go beyond the mere polymerization of fibrinogen into a fibrin network. The complex composition and localization of in vivo fibrin clots demonstrate the interplay between fibrin and/or fibrinogen and blood cells. Studying these protein–cell interactions and clot mechanical properties may represent new methods for the evaluation of cardiovascular diseases (the leading cause of death worldwide), creating new possibilities for clinical diagnosis, prognosis, and therapy. Expected final online publication date for the Annual Review of Biophysics, Volume 51 is May 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Lyophilised Platelet-Rich Fibrin: Physical and Biological Characterisation

Background: Platelet-rich fibrin (PRF) has gained popularity in craniofacial surgery, as it provides an excellent reservoir of autologous growth factors (GFs) that are essential for bone regeneration. However, the low elastic modulus, short-term clinical application, poor storage potential and limitations in emergency therapy use restrict its more widespread clinical application. This study fabricates lyophilised PRF (Ly-PRF), evaluates its physical and biological properties, and explores its application for craniofacial tissue engineering purposes. Material and methods: A lyophilisation method was applied, and the outcome was evaluated and compared with traditionally prepared PRF. We investigated how lyophilisation affected PRF’s physical characteristics and biological properties by determining: (1) the physical and morphological architecture of Ly-PRF using SEM, and (2) the kinetic release of PDGF-AB using ELISA. Results: Ly-PRF exhibited a dense and homogeneous interconnected 3D fibrin network. Moreover, clusters of morphologically consistent cells of platelets and leukocytes were apparent within Ly-PRF, along with evidence of PDGF-AB release in accordance with previously reports. Conclusions: The protocol established in this study for Ly-PRF preparation demonstrated versatility, and provides a biomaterial with growth factor release for potential use as a craniofacial bioscaffold.

Does Fibrin Structure Contribute to the Increased Risk of Thrombosis in COVID-19 ICU Patients?

Introduction: SARS-CoV-2 is responsible for a global pandemic, with almost 200 million confirmed cases. SARS-CoV-2 infection can lead to various disease states, from only mild symptoms in the majority of cases to severe disease, which is associated with an increased incidence of venous thromboembolism (VTE). We hypothesized that an altered fibrin network structure contributes to VTE in COVID-19 patients by affecting thrombus stability and fibrinolysis sensitivity. By studying the fibrin network of COVID-19 patients, we aimed to unravel the mechanisms that contribute to the increased risk of VTE in COVID-19 patients. Methods: Between April 2020 and December 2020, we collected plasma samples from patients with COVID-19 admitted to the intensive care unit (ICU) of the Erasmus Medical Center. We included patients with confirmed VTE diagnosed on CT-angiography, and COVID-19 patients without confirmed VTE during ICU admission. Samples were collected on admission to the ICU and after confirmed VTE or at similar time points in ICU patients without confirmed VTE. In addition, we collected plasma from COVID-19 patients at admission to general wards without confirmed VTE and from healthy controls. Clots were formed by mixing citrated plasma with thrombin (final concentration 1 U/ml) and calcium (17 mM). We imaged the clots using stimulated emission depletion (STED) microscopy, a super-resolution technique in which a depletion laser is used to selectively switch off fluorophores surrounding the focal point, thereby increasing the resolution. In these images, fibrin fiber diameters were measured using the Local Thickness plugin of ImageJ. Fiber density was quantified as percentage of area in Z-stacks of confocal microscopy images. Finally, a clot lysis assay based on turbidity was used to determine sensitivity to fibrinolysis (clot lysis time) and clot density (difference between maximum and baseline absorbance). Differences in fibrin network properties between groups were tested using One-Way ANOVA with Bonferroni post-hoc tests and linear regression with and without adjustment for fibrinogen levels. Results: We included 21 COVID-19 ICU patients with confirmed VTE, 20 COVID-19 ICU patients without confirmed VTE, 10 COVID-19 ward patients and 7 healthy controls. Mean age was comparable between the groups, while BMI was higher in COVID-19 patients than in healthy controls (Table 1). Levels of fibrinogen, D-dimer and anti-Xa were significantly higher in COVID-19 ICU patients than in COVID-19 ward patients and healthy controls. FVIII levels were significantly higher in COVID-19 ICU patients than in healthy controls, while FXIII levels were significantly lower. On admission to the ICU, clot density was significantly higher in COVID-19 ICU patients with and without confirmed VTE than in healthy controls (Figure 1 and Table 2). However, after adjustment for fibrinogen levels, this difference disappears. Clot lysis time was significantly longer in clots from COVID-19 ICU patients than in clots from healthy controls, regardless of fibrinogen levels (Table 2). COVID-19 ICU patients with confirmed VTE also showed a significant longer clot lysis time than COVID-19 ward patients. Interestingly, in the clot lysis assay, fibrinolysis did not occur in 25% of COVID-19 ICU patients with VTE versus 9.5% of COVID-19 ICU patients without VTE (Figure 2). This fibrinolysis shutdown was never observed in clots from healthy controls and COVID-19 ward patients. Fibrin fiber diameters were comparable between the groups. In the clots from plasma samples collected at admission to the ICU, there were no differences between COVID-19 ICU patients with and without VTE (Figure 2). However, when comparing clots prepared from plasma collected at the second time point (after VTE or at a similar time point for patients without VTE), we observed significant longer clot lysis times in patients with confirmed VTE (97.4 [88.5-158.8] min) than in patients without confirmed VTE (80.0 [76.0-97.8] min) (p=0.03). Finally, there were no significant changes between clots from plasma before and after VTE or between the two time points in patients without VTE, except for a decreased clot lysis time over time for COVID-19 ICU patients without confirmed VTE. Conclusion: Our results suggest that SARS-CoV-2 infection increases clot density and decreases clot susceptibility to fibrinolysis, and that these changes relate to the severity of the disease. Figure 1 Figure 1. Disclosures Kruip: Daiichi Sankyo: Research Funding; Bayer: Honoraria, Research Funding.

Development of a mesoscopic framework spanning nanoscale protofibril dynamics to macro-scale fibrin clot formation

Thrombi form a micro-scale fibrin network consisting of an interlinked structure of nanoscale protofibrils, resulting in haemostasis. It is theorized that the mechanical effect of the fibrin clot is caused by the polymeric protofibrils between crosslinks, or to their dynamics on a nanoscale order. Despite a number of studies, however, it is still unknown, how the nanoscale protofibril dynamics affect the formation of the macro-scale fibrin clot and thus its mechanical properties. A mesoscopic framework would be useful to tackle this multi-scale problem, but it has not yet been established. We thus propose a minimal mesoscopic model for protofibrils based on Brownian dynamics, and performed numerical simulations of protofibril aggregation. We also performed stretch tests of polymeric protofibrils to quantify the elasticity of fibrin clots. Our model results successfully captured the conformational properties of aggregated protofibrils, e.g., strain-hardening response. Furthermore, the results suggest that the bending stiffness of individual protofibrils increases to resist extension.

Fibrin Network Porosity and Endo-/Exogenous Thrombin Cross-talk

The earliest assessment of fibrin network porosity used a liquid permeation system and confocal 3D microscopy, which was later replaced by scanning electron microscopy. Although the methods have extensively been applied in studies of health or disease, there remains debate on the choice of a proper clotting trigger. In this review, we assess published data and convey our opinions with regard to several issues. First, when the coagulation process is initiated by recombinant tissue factor (rTF) and phospholipids, the fibrin network porosity is regulated by the endogenous thrombin based on enzymatic activations of multiple coagulants. If purified thrombin (1.0 IU/mL) is employed as the clotting trigger, fibrin network porosity may be affected by exogenous thrombin, which directly polymerizes fibrinogen in plasma, and additionally by endogenous thrombin stemming from a “positive feedback loop” action of the added thrombin. Second, with use of either endogenous or exogenous thrombin, the concentration and clotting property of available fibrinogen both influence the fibrin network porosity. Third, in the assay systems in vitro, exogenous thrombin but not rTF-induced endogenous thrombin seems to be functional enough to activate factor XIII, which then contributes to a decrease in the fibrin network porosity. Fourth, fibrin network porosity determines the transport of fibrinolytic components into/through the clots and therefore serves as an indicator of the fibrinolysis potential in plasma.

Interaction between Different Implant Surfaces and Liquid Fibrinogen: A Pilot In Vitro Experiment

Background. Platelet concentrates like leucocyte- and platelet-rich fibrin (L-PRF) have been widely evaluated in different oral surgical procedures to promote the healing process. However, liquid L-PRF products such as liquid fibrinogen have been poorly explored, especially in the biomimetic functionalization of dental implants. The aim of this in vitro study is to evaluate the interaction between 5 different dental implant surfaces and liquid fibrinogen. Methods. Five commercially available dental implants with different surfaces (Osseospeed™, TiUnite™, SLActive®, Ossean®, and Plenum®) were immersed for 60 minutes in liquid fibrinogen obtained from healthy donors. After this period, the implants were removed and fixed for scanning electron microscopy (SEM). Results. All dental implants were covered by a fibrin mesh. However, noticeable noncontact areas were observed for the Osseospeed™, TiUnite™, and SLActive® surfaces. On the other hand, Ossean® and Plenum® surfaces showed a dense and uniform layer of fibrin covering almost the entire implant surface. The Osseospeed™, TiUnite™, and SLActive® surfaces presented with lower blood cell numbers inside the fibrin mesh compared with the others. Moreover, at higher magnification, thicker fibrin fibers were observed in contact with Ossean® and Plenum® surfaces. The Plenum ®surface showed the thickest fibers which also inserted and interconnect to the microroughness. Conclusion. The initial contact between an implant surface and the fibrin network differs significantly among different implant brands. Further studies are necessary to explore the clinical impact of these observations in the osseointegration process of dental implants.

Fibrin Clot Formation under Oxidative Stress Conditions

During coagulation, the soluble fibrinogen is converted into insoluble fibrin. Fibrinogen is a multifunctional plasma protein, which is essential for hemostasis. Various oxidative posttranslational modifications influence fibrinogen structure as well as interactions between various partners in the coagulation process. The aim was to examine the effects of oxidative stress conditions on fibrin clot formation in arterial atherothrombotic disorders. We studied the changes in in vitro fibrin network formation in three groups of patients—with acute coronary syndrome (ACS), with significant carotid artery stenosis (SCAS), and with acute ischemic stroke (AIS), as well as a control group. The level of oxidative stress marker malondialdehyde measured by LC-MS/MS was higher in SCAS and AIS patients compared with controls. Turbidic methods revealed a higher final optical density and a prolonged lysis time in the clots of these patients. Electron microscopy was used to visualize changes in the in vitro-formed fibrin network. Fibers from patients with AIS were significantly thicker in comparison with control and ACS fibers. The number of fibrin fibers in patients with AIS was significantly lower in comparison with ACS and control groups. Thus, oxidative stress-mediated changes in fibrin clot formation, structure and dissolution may affect the effectiveness of thrombolytic therapy.

Improving Fibrin Hydrogels’ Mechanical Properties, through Addition of Silica or Chitosan-Silica Materials, for Potential Application as Wound Dressings

Fibrin is a protein-based hydrogel formed during blood coagulation. It can also be produced in vitro from human blood plasma, and it is capable of resisting high deformations. However, after each deformation process, it loses high amounts of water, which subsequently makes it mechanically unstable and, finally, difficult to manipulate. The objective of this work was to overcome the in vitro fibrin mechanical instability. The strategy consists of adding silica or chitosan-silica materials and comparing how the different materials electrokinetic-surface properties affect the achieved improvement. The siliceous materials electrostatic and steric stabilization mechanisms, together with plasma protein adsorption on their surfaces, were corroborated by DLS and ζ-potential measurements before fibrin gelling. These properties avoid phase separation, favoring homogeneous incorporation of the solid into the forming fibrin network. Young’s modulus of modified fibrin hydrogels was evaluated by AFM to quantitatively measure stiffness. It increased 2.5 times with the addition of 4 mg/mL silica. A similar improvement was achieved with only 0.7 mg/mL chitosan-silica, which highlighted the contribution of hydrophilic chitosan chains to fibrinogen crosslinking. Moreover, these chains avoided the fibroblast growth inhibition onto modified fibrin hydrogels 3D culture observed with silica. In conclusion, 0.7 mg/mL chitosan-silica improved the mechanical stability of fibrin hydrogels with low risks of cytotoxicity. This easy-to-manipulate modified fibrin hydrogel makes it suitable as a wound dressing biomaterial.

Regenerative medicine: characterization of human bone matrix gelatin (BMG) and folded platelet-rich fibrin (F-PRF) membranes alone and in combination (sticky bone)

During the last two decades autologous platelet and leukocyte rich products (PRP; PRF), opened new perspectives in regenerative medicine. In particular regenerative dentistry played a pioneer role in the application of these products in bone regenerative cases. Many aspects of cytokines, such as, growth factor release, blood cell content and its characterization were reported, but some practical questions are still unanswered in the preparation of PRF membranes and sticky bones. A new folding technique was introduced that created a good quality, pliable, and strong F-PRF membrane with a dense fibrin network and more homogenous blood cell distribution. F-PRF produced a very promising sticky bone combined with human freeze-dried cortical bone matrix gelatin (BMG). There hasn’t been much focus on the quality and character of the applied bone and the optimal membrane/bone particle ratio has not been reported. A 0.125 g BMG/ml plasma (1 g/8 ml) seems like the ideal combination with maximal BMG adhesion capacity of the membrane. Particle distribution of BMG showed that 3/4 of the particles ranged between 300–1000 µ, the remnant 1/4 was smaller than 300 µ. The whole F-PRF membrane and its parts were compared with conventional A-PRF membrane concerning their resistance against proteolytic digestion. The F-PRF was superior to A-PRF, which dissolved within 4–5 days, while F-PRF was destroyed only after 11 days, so this provides a better chance for local bone morphogenesis. The F-PRF pieces had similar resistance to the whole intact one, so they can be ideal for surgical procedures without risk of fast disintegration.