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When formed in vivo, murine hemostatic thrombi exhibit a heterogeneous architecture comprised of distinct regions of densely and sparsely packed platelets. In this study, we utilize high-resolution electron microscopy alongside machine learning and physics-based simulations to investigate how such clot microstructure impacts molecular diffusivity. We used Serial Block Face - Scanning Electron Microscopy (SBF-SEM) to image select volumes of hemostatic masses formed in a mouse jugular vein, producing large stacks of high-resolution 2D images. Images were segmented using machine learning software (Cellpose), whose training was augmented by manually segmented images. The segmented images were then utilized as a computational domain for Lattice Kinetic Monte-Carlo (LKMC) simulations. This process constitutes a computational pipeline that combines purely data-derived biological domains with physics-driven simulations to estimate how molecular movement is hindered in a hemostatic platelet mass. Using our pipeline, we estimated that the hindered diffusion rates of a globular protein range from 2% to 40% of the unhindered rate, with denser packing regions lending to lower molecular diffusivity. These data suggest that coagulation reactions rates, thrombin generation and activity, as well as platelet releasate activity may be drastically impacted by the internal geometry of a hemostatic thrombus.

Microfluidic lab-on-a-chip technologies enable the analysis and manipulation of small fluid volumes and particles at small scales and the control of fluid flow and transport processes at the microscale, leading to the development of new methods to address a broad range of scientific and medical challenges. Microfluidic and lab-on-a-chip technologies have made a noteworthy impact in basic, preclinical, and clinical research, especially in hematology and vascular biology due to the inherent ability of microfluidics to mimic physiologic flow conditions in blood vessels and capillaries. With the potential to significantly impact translational research and clinical diagnostics, technical issues and incentive mismatches have stymied microfluidics from fulfilling this promise. We describe how accessibility, usability, and manufacturability of microfluidic technologies should be improved and how a shift in mindset and incentives within the field is also needed to address these issues. In this report, we discuss the state of the microfluidic field regarding current limitations and propose future directions and new approaches for the field to advance microfluidic technologies closer to translation and clinical use. While our report focuses on using blood as the prototypical biofluid sample, the proposed ideas and research directions can be extrapolated to other areas of hematology, oncology, biology, and medicine.

Background: During clotting, thrombin generates fibrin monomers and activates plasma-derived transglutaminase factor (F) XIIIa; collagen and thrombin-activated platelets offer thrombin-independent cellular FXIIIa (cFXIIIa) for clotting. Detecting fibrin on collagen and tissue factor surfaces in whole blood clotting typically uses complex reagents like fluorescent fibrinogen or antifibrin antibody., Objectives: We want to test whether the peptide using the α2- antiplasmin crosslinking mechanism by FXIIIa is a useful tool in both monitoring FXIIIa activity, and visualize and monitor fibrin formation, deposition, and extent of crosslinking within fibrin structures in whole blood clots formed under flow., Methods: We tested a fluorescent peptide derived from α2-antiplasmin sequence (Ac-GNQEQVSPLTLLKWC-fluorescein) to monitor the location of transglutaminase activity and fibrin during whole blood clotting under microfluidic flow (wall shear rate, 100 s -1 )., Results: The peptide rapidly colocated with accumulating fibrin due to transglutaminase activity, confirmed by Phe-Pro-Arg-chloromethylketone inhibiting fibrin and peptide labeling. The FXIIIa inhibitor T101 had no effect on fibrin generation but ablated the labeling of fibrin by the peptide. Similarly, Gly-Pro-Arg-Pro abated fibrin formation and thus strongly attenuated the peptide signal. At arterial wall shear rate (1000 s -1 ), less fibrin was formed, and consequently, less peptide labeling of fibrin was detected compared with venous conditions. The addition of tissue plasminogen activator caused a reduction of both fibrin and peptide signals. Also, the peptide strongly colocalized with fibrin (but not platelets) in clots from laser-injured mouse cremaster arterioles. For clotting under flow, FXIIIa activity was most likely plasma-derived since a RhoA inhibitor did not block α2-antiplasmin fragment cross-linking to fibrin., Conclusion: Under flow, the majority of FXIIIa-dependent fibrin labeling with peptide during clotting was distal of thrombin activity. The synthetic peptide provided a strong and sustained labeling of fibrin as it formed under flow., (© 2023 The Author(s).)

During clotting under flow, thrombin rapidly generates fibrin, whereas fibrin potently sequesters thrombin. This co-regulation was studied using microfluidic whole blood clotting on collagen/tissue factor, followed by buffer wash, and a start/stop cycling flow assay using the thrombin fluorogenic substrate, Boc-Val-Pro-Arg-AMC. After 3 min of clotting (100 s -1 ) and 5 min of buffer wash, non-elutable thrombin activity was easily detected during cycles of flow cessation. Non-elutable thrombin was similarly detected in plasma clots or arterial whole blood clots (1000 s -1 ). This thrombin activity was ablated by Phe-Pro-Arg-chloromethylketone (PPACK), apixaban, or Gly-Pro-Arg-Pro to inhibit fibrin. Reaction-diffusion simulations predicted 108 nM thrombin within the clot. Heparin addition to the start/stop assay had little effect on fibrin-bound thrombin, whereas addition of heparin-antithrombin (AT) required over 6 min to inhibit the thrombin, indicating a substantial diffusion limitation. In contrast, heparin-AT rapidly inhibited thrombin within microfluidic plasma clots, indicating marked differences in fibrin structure and functionality between plasma clots and whole blood clots. Addition of GPVI-Fab to blood before venous or arterial clotting (200 or 1000 s -1 ) markedly reduced fibrin-bound thrombin, whereas GPVI-Fab addition after 90 s of clotting had no effect. Perfusion of AF647-fibrinogen over washed fluorescein isothiocyanate (FITC)-fibrin clots resulted in an intense red layer around, but not within, the original FITC-fibrin. Similarly, introduction of plasma/AF647-fibrinogen generated substantial red fibrin masses that did not penetrate the original green clots, demonstrating that fibrin cannot be re-clotted with fibrinogen. Overall, thrombin within fibrin is non-elutable, easily accessed by peptides, slowly accessed by average-sized proteins (heparin/AT), and not accessible to fresh fibrinogen., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Platelets have toll-like receptors (TLRs); however, their function in thrombosis or hemostasis under flow conditions is not fully known. Thrombin-inhibited anticoagulated whole blood was treated with various TLR agonists and then perfused over fibrillar collagen using microfluidic assay at venous wall shear rate (100 s -1 ). Platelet deposition was imaged with fluorescent anti-CD61. For perfusion of whole blood without TLR agonist addition, platelets rapidly accumulated on collagen and eventually occluded the microchannels. Interestingly, most of the tested TLR agonists (Pam 3 CKS 4 , MALP-2, polyinosinic-polycytidylic acid HMW, imiquimod, and CpG oligodeoxynucleotides) strongly reduced platelet deposition on collagen, while only the TLR4 agonist endotoxin lipopolysaccharide (LPS) enhanced deposition. Following 90 sec of deposition under flow of untreated blood, the addition of various TLR-7 agonists (imiquimod, vesatolimod, and GSK2245035) all caused immediate blockade of further platelet deposition. Since TLR signaling can activate nuclear factor-kappaB (NF- κ B), the IKK-inhibitor (IKK inhibitor VII) and NF- κ B inhibitor (Bay 11-7082) were tested. The IKK/NF- κ B inhibitors strongly inhibited platelet deposition under flow. Furthermore, addition of Pam3CSK4 (TLR1/2 ligand), MALP-2 (TLR2/6 ligand), and Imquimod (TLR7 ligand) reduced phosphotidylserine (PS) exposure. Activation of TLR1/2, TLR2/6, TLR3, TLR7, and TLR9 in whole blood reduced platelet deposition under flow on collagen; however, LPS (major Gram negative bacterial pathogenic component) activation of LTR4 was clearly prothrombotic., Competing Interests: The authors state that they have no conflict of interest., (Copyright © 2023 Y. Liu and S. L. Diamond.)

The collagen receptor glycoprotein VI (GPVI) drives strong platelet activation, however its role at later stages of clotting remains less clear. Controlled timing of addition of anti-human GPVI Fab (clone E12) with microfluidic venous whole blood flow over collagen (± lipidated tissue factor, TF) produced distinct effects on platelets, fibrin, P-selectin exposure, and phosphatidylserine (PS) exposure. On collagen alone, Fab present initially potently reduced platelet deposition on collagen, while Fab added 90 s after initial platelet deposition, stopped subsequent platelet accumulation (despite the absence of fibrin). With thrombin generation via TF, Fab added at either t = 0 or 90 s had no effect on platelet deposition. However, Fab added initially, but not at 90-s, blocked fibrin formation. Gly-Pro-Arg-Pro ablated fibrin formation without effect on platelet accumulation (regardless of Fab added at t = 0 or 90 s), indicating thrombin signaling can suffice over GPVI signaling. Still, Fab moderately reduced P-selectin exposure with thrombin present and fibrin absent. On collagen/TF, Fab present initially ablated PS exposure, but had no effect when added 30 to 90-s later. The thrombin generated via PS exposure had an important role in driving platelet deposition in the presence of Fab, since inhibition of PS via annexin V binding in the presence of Fab significantly inhibited platelet deposition. We conclude GPVI signaling in the first platelet layer on collagen dictates thrombin and fibrin production, but the role of GPVI at subsequent times after formation of the first monolayer is obscured by thrombin-induced signaling., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Platelet contractility drives clot contraction to enhance clot density and stability. Clot contraction is typically studied under static conditions, with fewer studies of wall-adherent platelet clots formed under flow. We tested the effect of inhibitors of ADP and/or thromboxane A2 (TXA2) signaling on clot contraction. Using an eight-channel microfluidic device, we perfused PPACK-treated whole blood (WB) ± acetylsalicylic acid (ASA), 2-methylthioAMP (2-MeSAMP), and/or MRS-2179 over collagen (100/s) for 7.5 min, then stopped flow to observe contraction for 7.5 minutes. Two automated imaging methods scored fluorescent platelet percent contraction over the no-flow observation period: (1) "global" measurement of clot length and (2) "local" changes in surface area coverage of the numerous platelet aggregates within the clot. Total platelet fluorescence intensity (FI) decreased with concomitant decrease in global aggregate contraction when ASA, 2-MeSAMP, and/or MRS-2179 were present. Total platelet FI and global aggregate contraction were highly correlated ( R 2  = 0.87). In contrast, local aggregate contraction was more pronounced than global aggregate contraction across all inhibition conditions. However, ASA significantly reduced local aggregate contraction relative to conditions without TXA2 inhibition. P-selectin display was significantly reduced by ADP and TXA2 inhibition, but there was limited detection of global or local aggregate contraction in P-selectin-positive platelets across all conditions, as expected for densely packed "core" platelets. Our results demonstrate that global aggregate contraction is inhibited by ASA, 2-MeSAMP, and MRS-2179, while ASA more potently inhibited local aggregate contraction. These results help resolve how different platelet antagonists affect global and local clot structure and function., Competing Interests: Conflict of Interest None declared., (The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution License, permitting unrestricted use, distribution, and reproduction so long as the original work is properly cited. ( https://creativecommons.org/licenses/by/4.0/ ).)

Purpose: Coronary flow is affected by evolving events such as atherosclerotic plaque formation, rupture, and thrombosis, resulting in myocardial ischemia and infarction. Highly resolved 3D hemodynamic data at the stenosis is essential to model shear-sensitive thrombotic events in coronary artery disease., Methods: We developed a hybrid 1D-3D simulation framework to compute patient-specific coronary hemodynamics efficiently. A 1D model of the coronary flow is coupled to an image-based 3D model of the region of interest. This framework affords the advantages of reduced-order modeling, decreasing the global computational cost, without sacrificing the accuracy of the quantities of interest., Results: We validated our 1D-3D model against full 3D coronary simulations in healthy and diseased conditions. Our results showed good agreement between the 3D and the 1D-3D models while reducing the computational cost by 40-fold compared to the 3D simulation. The 1D-3D model predicted left/right coronary flow distribution within 3% and provided an accurate estimation of fractional flow reserve and wall shear stress distribution at the stenosis comparable to the 3D simulation., Conclusion: Savings in computational cost may be significant in situations with changing geometry, such as growing thrombosis. Also, this approach would allow quantifying the time-dependent effect of thrombotic growth and occlusion on the global coronary circulation., (© 2021. Biomedical Engineering Society.)

Modeling thrombus growth in pathological flows allows evaluation of risk under patient-specific pharmacological, hematological, and hemodynamical conditions. We have developed a 3D multiscale framework for the prediction of thrombus growth under flow on a spatially resolved surface presenting collagen and tissue factor (TF). The multiscale framework is composed of four coupled modules: a Neural Network (NN) that accounts for platelet signaling, a Lattice Kinetic Monte Carlo (LKMC) simulation for tracking platelet positions, a Finite Volume Method (FVM) simulator for solving convection-diffusion-reaction equations describing agonist release and transport, and a Lattice Boltzmann (LB) flow solver for computing the blood flow field over the growing thrombus. A reduced model of the coagulation cascade was embedded into the framework to account for TF-driven thrombin production. The 3D model was first tested against in vitro microfluidics experiments of whole blood perfusion with various antiplatelet agents targeting COX-1, P2Y1, or the IP receptor. The model was able to accurately capture the evolution and morphology of the growing thrombus. Certain problems of 2D models for thrombus growth (artifactual dendritic growth) were naturally avoided with realistic trajectories of platelets in 3D flow. The generalizability of the 3D multiscale solver enabled simulations of important clinical situations, such as cylindrical blood vessels and acute flow narrowing (stenosis). Enhanced platelet-platelet bonding at pathologically high shear rates (e.g., von Willebrand factor unfolding) was required for accurately describing thrombus growth in stenotic flows. Overall, the approach allows consideration of patient-specific platelet signaling and vascular geometry for the prediction of thrombotic episodes., Competing Interests: The authors have declared that no competing interests exist.

Risk quantification algorithms in the ICU can provide (1) an early alert to the clinician that a patient is at extreme risk and (2) help manage limited resources efficiently or remotely. With electronic health records, large data sets allow the training of predictive models to quantify patient risk. A gradient boosting classifier was trained to predict high-risk and low-risk trauma patients, where patients were labeled high-risk if they expired within the next 10 hours or within the last 10% of their ICU stay duration. The MIMIC-III database was filtered to extract 5,400 trauma patient records (526 non-survivors) each of which contained 5 static variables (age, gender, etc.) and 28 dynamic variables (e.g., vital signs and metabolic panel). Training data was also extracted from the dynamic variables using a 3-hour moving time window whereby each window was treated as a unique patient-time fragment. We extracted the mean, standard deviation, and skew from each of these 3-hour fragments and included them as inputs for training. Additionally, a survival metric upon admission was calculated for each patient using a previously developed National Trauma Data Bank (NTDB)-trained gradient booster model. The final model was able to distinguish between high-risk and low-risk patients to an AUROC of 92.9%, defined as the area under the receiver operator characteristic curve. Importantly, the dynamic survival probability plots for patients who die appear considerably different from those who survive, an example of reducing the high dimensionality of the patient record to a single trauma trajectory., Competing Interests: The authors have declared that no competing interests exist.

A highly reduced extrinsic pathway coagulation model (8 ODEs) under flow considered a thin 15-micron platelet layer where transport limitations were largely negligible (except for fibrinogen) and where cofactors (FVIIa, FV, FVIII) were not rate-limiting. By including thrombin feedback activation of FXI and the antithrombin-I activities of fibrin, the model accurately simulated measured fibrin formation and thrombin fluxes. Using this reduced model, we conducted 10,000 Monte Carlo (MC) simulations for ±50% variation of 5 plasma zymogens and 2 fibrin binding sites for thrombin. A sensitivity analysis of zymogen concentrations indicated that FIX activity most influenced thrombin generation, a result expected from hemophilia A and B. Averaging all MC simulations confirmed both the mean and standard deviation of measured fibrin generation on 1 tissue factor (TF) molecule per μm2. Across all simulations, free thrombin in the layer ranged from 20 to 300 nM (mean: 50 nM). The top 2% of simulations that produced maximal fibrin were dominated by conditions with low antithrombin-I activity (decreased weak and strong sites) and high FIX concentration. In contrast, the bottom 2% of simulations that produced minimal fibrin were dominated by low FIX and FX. The percent reduction of fibrin by an ideal FXIa inhibitor (FXI = 0) ranged from 71% fibrin reduction in the top 2% of MC simulations to only 34% fibrin reduction in the bottom 2% of MC simulations. Thus, the antithrombotic potency of FXIa inhibitors may vary depending on normal ranges of zymogen concentrations. This reduced model allowed efficient multivariable sensitivity analysis., Competing Interests: The authors have declared that no competing interests exist.

Point-of-care diagnostics of platelet and coagulation function present demanding challenges. Current clinical diagnostics often use centrifuged plasmas or platelets and frozen plasma standards, recombinant protein standards, or even venoms. Almost all commercialized tests of blood do not recreate the in vivo hemodynamics where platelets accumulate to high densities and thrombin is generated from a procoagulant surface. Despite numerous drugs that target platelets, insufficient coagulation, or excess coagulation, POC blood testing is essentially limited to viscoelastic methods that provide a clotting time, clot strength, and clot lysis, while used mostly in trauma centers with specialized capabilities. Microfluidics now allows small volumes of whole blood (<1 mL) to be tested under venous or arterial shear rates with multi-color readouts to follow platelet function, thrombin generation, fibrin production, and clot stability. Injection molded chips containing pre-patterned fibrillar collagen and lipidated tissue factor can be stored dry for 6 months at 4C, thus allowing rapid blood testing on single-use disposable chips. Using only a small imaging microscope and micropump, these microfluidic devices can detect platelet inhibitors, direct oral anticoagulants (DOACs) and their reversal agents. POC microfluidics are ideal for neonatal surgical applications that involve small blood samples, rapid DOAC testing in stroke or bleeding or emergency surgery situations with patients presenting high risk cofactors for either bleeding or thrombosis.

Distinct from dilute, isotropic, and homogeneous reaction systems typically used in laboratory kinetic assays, blood is concentrated, two-phase, flowing, and highly anisotropic when clotting on a surface. This review focuses on spatial gradients that are generated and can dictate thrombus structure and function. Novel experimental and computational tools have recently emerged to explore reaction-transport coupling during clotting. Multiscale simulations help bridge tissue length scales (the coronary arteries) to millimeter scales of a growing clot to the microscopic scale of single-cell signaling and adhesion. Microfluidic devices help create and control pathological velocity profiles, albeit at a low Reynolds number. Since rate processes and force loading are often coupled, this review highlights prevailing convective-diffusive transport physics that modulate cellular and molecular processes during thrombus formation., Competing Interests: Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Many biologically important cell binding processes, such as the rolling of leukocytes in the vasculature, are multivalent, being mediated by large numbers of weak binding ligands. Quantitative agreement between experiments and models of rolling has been elusive and often limited by the poor understanding of the binding and unbinding kinetics of the ligands involved. Here, we present a cell-free experimental model for such rolling, consisting of polymer microspheres whose adhesion to a glass surface is mediated by ligands with well-understood force-dependent binding free energy-short complementary DNA strands. We observe robust rolling activity for certain values of the shear rate and the grafted DNA strands' binding free energy and force sensitivity. The simulation framework developed to model leukocyte rolling, adhesive dynamics, quantitatively captures the mean rolling velocity and lateral diffusivity of the experimental particles using known values of the experimental parameters. Moreover, our model captures the velocity variations seen within the trajectories of single particles. Particle-to-particle variations can be attributed to small, plausible differences in particle characteristics. Overall, our findings confirm that state-of-the-art adhesive dynamics simulations are able to capture the complex physics of particle rolling, boding well for their extension to modeling more complex systems of rolling cells., (Copyright © 2021 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The development of anti-drug antibodies (ADAs) is a serious outcome of treatment strategies involving biological medicines. Coagulation factor VIII (FVIII) is used to treat haemophilia A patients, but its immunogenicity precludes a third of severe haemophiliac patients from receiving this treatment. The availability of patient-derived anti-drug antibodies can help us better understand drug immunogenicity and identify ways to overcome it. Thus, there were two aims to this work: (i) to develop and characterise a panel of recombinant, patient-derived, monoclonal antibodies covering a range of FVIII epitopes with varying potencies, kinetics and mechanism of action, and (ii) to demonstrate their applicability to assay development, evaluation of FVIII molecules and basic research. For the first objective we used recombinant antibodies to develop a rapid, sensitive, flexible and reproducible ex vivo assay that recapitulates inhibitor patient blood using blood from healthy volunteers. We also demonstrate how the panel can provide important information about the efficacy of FVIII products and reagents without the need for patient or animal material. These materials can be used as experimental exemplars or controls, as well as tools for rational, hypothesis-driven research and assay development in relation to FVIII immunogenicity and FVIII-related products., (© 2021 British Society for Haematology and John Wiley & Sons Ltd.)

Background: Thrombin activates fibrinogen and binds the fibrin E-domain (K d  ~ 2.8 μM) and the splice variant γ'-domain (K d  ~ 0.1 μM). We investigated if the loading of D-Phe-Pro-Arg-chloromethylketone inhibited thrombin (PPACK-thrombin) onto fibrin could enhance fibrin stability., Methods: A 384-well plate thermal shift assay (TSA) with SYPRO-orange provided melting temperatures (T m ) of thrombin, PPACK-thrombin, fibrinogen, fibrin monomer, and fibrin., Results: Large increases in T m indicated that calcium led to protein stabilization (0 vs. 2 mM Ca 2+ ) for fibrinogen (54.0 vs. 62.3 °C) and fibrin (62.3 vs. 72.2 °C). Additionally, active site inhibition with PPACK dramatically increased the T m of thrombin (58.3 vs. 78.3 °C). Treatment of fibrinogen with fibrin polymerization inhibitor GPRP increased fibrinogen stability by ΔT m  = 9.3 °C, similar to the ΔT m when fibrinogen was converted to fibrin monomer (ΔT m  = 8.8 °C) or to fibrin (ΔT m  = 10.4 °C). Addition of PPACK-thrombin at high 5:1 M ratio to fibrin(ogen) had little effect on fibrin(ogen) T m values, indicating that thrombin binding does not detectably stabilize fibrin via a putative bivalent E-domain to γ'-domain interaction., Conclusions: TSA was a sensitive assay of protein stability and detected: (1) the effects of calcium-stabilization, (2) thrombin active site labeling, (3) fibrinogen conversion to fibrin, and (4) GPRP induced changes in fibrinogen stability being essentially equivalent to that of fibrin monomer or polymerized fibrin., Significance: The low volume, high throughput assay has potential for use in understanding interactions with rare or mutant fibrin(ogen) variants., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background:  As thrombosis proceeds, certain platelets in a clot expose phosphatidylserine (PS) on their outer membrane. These PS + platelets subsequently sort to the perimeter of the mass via platelet contraction. It remains unclear how thrombin and fibrin may alter PS + platelet sorting within a clot., Objective:  We investigated the role of fibrin in PS + platelet sorting., Methods:  We used an 8-channel microfluidic assay of clotting over collagen (±tissue factor) at 100 s -1 initial wall shear rate. Temporal PS + platelet sorting was measured using a Pearson's correlation coefficient between the annexin V distribution in a clot at 9 versus 15 minutes. Spatial PS + platelet sorting was measured using an autocorrelation metric of the final annexin V distribution., Results:  By 6 minutes, PS + platelets were distributed throughout the platelet deposits and became highly spatially sorted by 15 minutes when thrombin and fibrin were blocked with Phe-Pro-Arg-chloromethylketone (PPACK). Fibrin polymerization (no PPACK) attenuated temporal and spatial PS sorting and clot contraction. With Gly-Pro-Arg-Pro (GPRP) added to block fibrin polymerization, PS sorting was prominent as was clot contraction. Exogenously added tissue plasminogen activator drove fibrinolysis that in turn promoted clot contraction and PS sorting, albeit to a lesser degree than the PPACK or GPRP conditions. Clots lacking fibrin displayed 3.6 times greater contraction than clots with fibrin., Conclusion:  PS sorting correlated with clot contraction, as previously reported. However, fibrin inversely correlated with both percent contraction and PS sorting. Fibrin attenuated clot contraction and PS sorting relative to clots without fibrin., Competing Interests: None declared., (Thieme. All rights reserved.)

A 400-estimator gradient boosting classifier was trained to predict survival probabilities of trauma patients. The National Trauma Data Bank (NTDB) provided 799233 complete patient records (778303 survivors and 20930 deaths) each containing 32 features, a number further reduced to only 8 features via the permutation importance method. Importantly, the 8 features can all be readily determined at admission: systolic blood pressure, heart rate, respiratory rate, temperature, oxygen saturation, gender, age and Glasgow coma score. Since death was rare, a rebalanced training set was used to train the model. The model is able to predict a survival probability for any trauma patient and accurately distinguish between a deceased and survived patient in 92.4% of all cases. Partial dependence curves (Psurvival vs. feature value) obtained from the trained model revealed the global importance of Glasgow coma score, age, and systolic blood pressure while pulse rate, respiratory rate, temperature, oxygen saturation, and gender had more subtle single variable influences. Shapley values, which measure the relative contribution of each of the 8 features to individual patient risk, were computed for several patients and were able to quantify patient-specific warning signs. Using the NTDB to sample across numerous patient traumas and hospital protocols, the trained model and Shapley values rapidly provides quantitative insight into which combination of variables in an 8-dimensional space contributed most to each trauma patient's predicted global risk of death upon emergency room admission., Competing Interests: The authors have declared that no competing interests exist.

Background: Platelet dysfunction often accompanies trauma-induced coagulopathy. Because soluble fibrin impairs platelet glycoprotein VI (GPVI) signaling and platelets of trauma patients can display impaired calcium mobilization, we explored the role of fibrinolysis on platelet dysfunction during trauma., Methods: Convulxin-induced GPVI calcium mobilization was investigated in healthy platelet-rich plasma (PRP) pretreated with thrombin and tissue plasminogen activator (tPA). Blood samples from healthy participants (n = 7) and trauma patients (n = 22) were tested for platelet calcium mobilization, plasma D-dimer, platelet D-dimer binding (via flow cytometry), and platelet lumi-aggregometry., Results: For healthy platelets, maximal platelet dysfunction was observed when cross-linked soluble fibrin (no tPA) or cross-linked fibrin degradation products (FDPs) were generated in suspension before convulxin stimulation. Lack of fibrin polymerization (inhibited by Gly-Pro-Arg-Pro [GPRP]) or lack of factor XIIIa cross-linking (T101-inhibited) restored GPVI signaling, whereas non-cross-linked FDPs only partially blocked signaling induced by convulxin. In addition, D-dimer added to healthy PRP impaired platelet aggregation and dense granule release induced by various agonists. Plasma D-dimer level was strongly correlated (R = 0.8236) with platelet dysfunction as measured by platelet calcium mobilization induced with various agonists. By 48 to 120 h after trauma, plasma D-dimer levels declined, and platelet function increased significantly but not to healthy levels. Trauma platelets displayed elevated D-dimer binding that was only partially reduced by αIIbβ3-inhibitor GR144053. After 60-minute incubation, washed healthy platelets resuspended in plasma from trauma patients captured approximately 10 000 D-dimer equivalents per platelet., Conclusions: During trauma, D-dimer and FDPs inhibit platelets, potentially via GPVI and integrin αIIbβ3 engagement, contributing to a fibrinolysis-dependent platelet loss-of-function phenotype., (© American Association for Clinical Chemistry 2020. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Custom polydimethylsiloxane (PDMS) microfluidic devices allow for small-volume human blood research under hemodynamic conditions of bleeding and clotting. However, issues of PDMS molding/assembly, bio-coating, and sample preparation often limit their point-of-care use. We aim to develop a microfluidic device that has the same utility as previously established PDMS devices but which is more usable in point-of-care operation. We designed an injection-molded 1 × 3 in. 2 device with eight flow paths crossing a bio-printed surface of a collagen/tissue factor. The device is rapidly primed and compatible with multi-channel pipetting (<0.5 ml blood) and operates under venous or arterial shear rates using constant flow rate or constant pressure modes. Platelet and fibrin deposition were monitored dynamically by the imaging of immunofluorescence. For whole blood clotting at a wall shear rate of 200 s -1 , the intrachip CV at 400 s for platelet and fibrin deposition was 10% and the interdonor CV at 400 s was 30% for platelet and 22% for fibrin deposition (across 10 healthy donors). No significant difference was detected for samples tested on a new chip vs a chip stored for 6 months at 4 °C. Using the fibrin signal, dose-response testing of whole blood revealed IC 50 's of 120 nM for rivaroxaban and apixaban, and 60 nM for dabigatran. A complete reversal of apixaban inhibition was observed for an equimolar addition of Xa DOAC reversal agent Andexanet Alfa. We demonstrate the ability to manufacture single-use, storage-stable eight-channel chips. In clinical settings, such chips may help evaluate patient bleeding risk, therapy choice, drug activity, or reversal., (Copyright © 2020 Author(s).)

Background: Hemostatic clots have a P-selectin positive platelet core covered with a shell of P-selectin negative platelets., Objective: To develop a new human blood microfluidic assay to interrogate core/shell mechanics., Methods: A 2-stage assay perfused whole blood over collagen/± tissue factor (TF) for 180 seconds at 100 s -1 wall shear rate, followed by buffer perfusion at either 100 s -1 (venous) or 1000 s -1 (arterial). This microfluidic assay used an extended channel height (120 µm), allowing buffer perfusion well before occlusion., Results: Clot growth on collagen stopped immediately with buffer exchange, revealing ~10% reduction in platelet fluorescence intensity (at 100 s -1 ) and ~30% (at 1000 s -1 ) by 1200 seconds. Thrombin generation (on collagen/TF) reduced erosion at either buffer flow rate. P-selectin-positive platelets were stable (no erosion) against 1000 s -1 , in contrast to P-selectin negative platelets. Thrombin inhibition (with D-Phe-Pro-Arg-CMK) reduced the number of P-selectin-positive platelets and lowered thrombus stability through the reduction of P-selectin-positive platelets. Interestingly, fibrin inhibition (with H-Gly-Pro-Arg-Pro-OH acetate salt) increased the number of P-selectin-positive platelets but did not lower stability, suggesting that fibrin was only in the core region. Thromboxane inhibition reduced P-selectin-positive platelets and caused a nearly 60% reduction of the clot at arterial buffer flow. P2Y1 antagonism reduced clot size and the number of P-selectin-positive platelets and reduced the stability of P-selectin-negative platelets., Conclusion: The 2-stage assay (extended channel height plus buffer exchange) interrogated platelet stability using human blood. Under all conditions, P-selectin-positive platelets never left the clot., (© 2020 The Authors. Research and Practice in Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis (ISTH).)

Kinase inhibitors can pose bleeding risks as platelet signaling evolves during clotting. Using microfluidics (200 s -1 wall shear rate) to perfuse Factor XIIa-inhibited or thrombin-inhibited whole blood (WB) over collagen ± tissue factor (TF), we explored the potency of the Src family kinase (SFK) inhibitor dasatinib or the spleen tyrosine kinase (Syk) inhibitor GS-9973 present at clot initiation or added after 90 s (via rapid switch to inhibitor-pretreated WB). When initially present, dasatinib potently inhibited platelet deposition on collagen (no TF). Furthermore, dasatinib immediately inhibited subsequent platelet deposition when introduced 90 s after clot initiation. However, when thrombin was generated, dasatinib was markedly less potent against platelet deposition on collagen/TF (but blocked fibrin deposition) and had no effect when added 90 s after clot initiation. Similarly, dasatinib added at 90 s had no effect on clotting on collagen/TF when fibrin was also blocked with Gly-Pro-Arg-Pro, indicating that strong thrombin-induced signaling (but not fibrin-induced signaling) can bypass the SFK inhibition at later times. The Syk inhibitor GS-9973 was less potent than dasatinib when present initially, but inhibited clot growth when added at 90 s, even in the presence of thrombin (±fibrin). Interestingly, the active form (R-406) of fostamatinib inhibits platelet function in only 2 0f 5 healthy blood samples. SFK-inhibitors may have reduced antithrombotic activity and reduced bleeding risks in settings of high TF and local thrombin generation. For oncology patients, SFK-inhibitors like dasatinib may have reduced antithrombotic activity and reduced bleeding risk in settings of local thrombin generation., Competing Interests: Declaration of competing interest The authors declare no potential conflicts of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The frequency and intensity of marine heatwaves that result in coral bleaching events have increased over recent decades and led to catastrophic losses of reef-building corals in many regions. The high-latitude coral assemblages at Lord Howe Island, which is a UNESCO listed site is the world southernmost coral community, were exposed to successive thermal anomalies following a fast phase-transition of the record-breaking 2009 to 2010 warm pool El Niño in the Central Pacific to a strong La Niña event in late 2010. The coral community experienced severe and unprecedented consecutive bleaching in both 2010 and 2011. Coral health surveys completed between March 2010 and September 2012 quantified the response and recovery of approximately 43,700 coral colonies to these successive marine heatwaves. In March 2010, coral bleaching ranged from severe, with 99% of colonies bleached at some shallow lagoon sites, to mild at deeper reef slope sites, with only 17% of individuals affected. Significant immediate mortality from thermal stress was evident during the peak of the bleaching event. Overall, species in the genera Pocillopora, Stylophora, Seriatopora and Porites were the most affected, while minimal bleaching and mortality was recorded among members of other coral families (e.g. Acroporidae, Dendrophyllidae & Merulinidae). Surviving corals underwent a subsequent, but much less intense, thermal anomaly in 2011 that led to a disproportionate bleaching response among susceptible taxa. While this observation indicates that the capacity of thermally susceptible high-latitude corals to acclimatize to future ocean warming may be limited, particularly if bleaching events occur annually, our long-term survey data shows that coral cover at most sites recovered to pre-bleaching levels within three years in the absence of further thermal anomalies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Crown Copyright © 2020. Published by Elsevier B.V. All rights reserved.)

Background: Microfluidic clotting assays permit drug action studies for hemophilia therapeutics under flow. However, limited availability of patient samples and Inter-donor variability limit the application of such assays, especially with many patients on prophylaxis., Objective: To develop approaches to phenocopy hemophilia using modified healthy blood in microfluidic assays., Methods: Corn trypsin inhibitor (4 µg/mL)-treated healthy blood was dosed with either anti-factor VIII (FVIII; hemophilia A model) or a recombinant factor IX (FIX) missense variant (FIX-V181T; hemophilia B model). Treated blood was perfused at 100 s -1 wall shear rate over collagen/tissue factor (TF) or collagen/factor XIa (FXIa)., Results: Anti-FVIII partially blocked fibrin production on collagen/TF, but completely blocked fibrin production on collagen/FXIa, a phenotype reversed with 1 µmol/L bispecific antibody (emicizumab), which binds FIXa and factor X. As expected, emicizumab had no significant effect on healthy blood (no anti-FVIII present) perfused over collagen/FXIa. The efficacy of emicizumab in anti-FVIII-treated healthy blood phenocopied the action of emicizumab in the blood of a patient with hemophilia A perfused over collagen/FXIa. Interestingly, a patient-derived FVIII-neutralizing antibody reduced fibrin production when added to healthy blood perfused over collagen/FXIa. For low TF surfaces, reFIX-V181T (50 µg/mL) fully blocked platelet and fibrin deposition, a phenotype fully reversed with anti-TFPI., Conclusion: Two new microfluidic hemophilia A and B models demonstrate the potency of anti-TF pathway inhibitor, emicizumab, and a patient-derived inhibitory antibody. Using collagen/FXIa-coated surfaces resulted in reliable and highly sensitive hemophilia models., (© 2019 The Authors. Research and Practice in Thrombosis and Haemostasis published by Wiley Periodicals, Inc on behalf of International Society on Thrombosis and Haemostasis.)

The ability of a blood clot to modulate blood flow is determined by the clot's resistance, which depends on its structural features. For a flow with arterial shear, we investigated the characteristic patterns relating to clot shape, size, and composition on the one hand, and its viscous resistance, intraclot axial flow velocity, and shear distributions on the other. We used microfluidic technology to measure the kinetics of platelet, thrombin, and fibrin accumulation at a thrombogenic surface coated with collagen and tissue factor (TF), the key clot-formation trigger. We subsequently utilized the obtained data to perform additional calibration and validation of a detailed computational fluid dynamics model of spatial clot growth under flow. We then ran model simulations to gain insights into the resistance of clots formed under our experimental conditions. We found that increased thrombogenic surface length and TF surface density enhanced the bulk thrombin and fibrin generation in a nonadditive, synergistic way. The height of the platelet deposition domain-and, therefore, clot occlusivity-was rather robust to thrombogenic surface length and TF density variations, but consistently increased with time. Clot viscous resistance was non-uniform and tended to be higher in the fibrin-rich, inner "core" region of the clot. Interestingly, despite intraclot structure and viscous resistance variations, intraclot flow velocity variations were minor compared to the abrupt decrease in flow velocity around the platelet deposition region. Our results shed new light on the connection between the structure of clots under arterial shear and spatiotemporal variations in their resistance to flow.

During thrombosis, thrombin generates fibrin, however fibrin reversibly binds thrombin with low affinity E-domain sites (KD = 2.8 μM) and high affinity γ'-fibrin sites (KD = 0.1 μM). For blood clotting on collagen/tissue factor (1 TF-molecule/μm2) at 200 s-1 wall shear rate, high μM-levels of intraclot thrombin suggest robust prothrombin penetration into clots. Setting intraclot zymogen concentrations to plasma levels (and neglecting cofactor rate limitations) allowed the linearization of 7 Michaelis-Menton reactions between 6 species to simulate intraclot generation of: Factors FXa (via TF/VIIa or FIXa), FIXa (via TF/FVIIa or FXIa), thrombin, fibrin, and FXIa. This reduced model [7 rates, 2 KD's, enzyme half-lives~1 min] predicted the measured clot elution rate of thrombin-antithrombin (TAT) and fragment F1.2 in the presence and absence of the fibrin inhibitor Gly-Pro-Arg-Pro. To predict intraclot fibrin reaching 30 mg/mL by 15 min, the model required fibrinogen penetration into the clot to be strongly diffusion-limited (actual rate/ideal rate = 0.05). The model required free thrombin in the clot (~100 nM) to have an elution half-life of ~2 sec, consistent with measured albumin elution, with most thrombin (>99%) being fibrin-bound. Thrombin-feedback activation of FXIa became prominent and reached 5 pM FXIa at >500 sec in the simulation, consistent with anti-FXIa experiments. In predicting intrathrombus thrombin and fibrin during 15-min microfluidic experiments, the model revealed "cascade amplification" from 30 pM levels of intrinsic tenase to 15 nM prothrombinase to 15 μM thrombin to 90 μM fibrin. Especially useful for multiscale simulation, this reduced model predicts thrombin and fibrin co-regulation during thrombosis under flow., Competing Interests: The authors have declared that no competing interests exist.

Quantifying the relationship between vascular injury and the dynamic bleeding rate requires a multiscale model that accounts for changing and coupled hemodynamics between the global and microvascular levels. A lumped, global hemodynamic model of the human cardiovascular system with baroreflex control was coupled to a local 24-level bifurcating vascular network that spanned diameters from the muscular artery scale (0.1-1.3 mm) to capillaries (5-10 μm) via conservation of momentum and conservation of mass boundary conditions. For defined injuries of severing all vessels at each n th-level, the changing pressures and flowrates were calculated using prescribed shear-dependent hemostatic clot growth rates (normal or coagulopathic). Key results were as follows: 1 ) the upstream vascular network rapidly depressurizes to reduce blood loss; 2 ) wall shear rates at the hemorrhaging wound exit are sufficiently high (~10,000 s -1 ) to drive von Willebrand factor unfolding; 3 ) full coagulopathy results in >2-liter blood loss in 2 h for severing all vessels of 0.13- to 0.005-mm diameter within the bifurcating network, whereas full hemostasis limits blood loss to <100 ml within 2 min; and 4 ) hemodilution from transcapillary refill increases blood loss and could be implicated in trauma-induced coagulopathy. A sensitivity analysis on length-to-diameter ratio and branching exponent demonstrated that bleeding was strongly dependent on these tissue-dependent network parameters. This is the first bleeding model that prescribes the geometry of the injury to calculate the rate of pressure-driven blood loss and local wall shear rate in the presence or absence of coagulopathic blood. NEW & NOTEWORTHY We developed a multiscale model that couples a lumped, global hemodynamic model of a patient to resolved, single-vessel wounds ranging from the small artery to capillary scale. The model is able to quantify wall shear rates, seal rates, and blood loss rates in the presence and absence of baroreflex control and hemodilution.

Neutrophils can release extracellular traps (NETs) in infectious, inflammatory and thrombotic diseases. NETs have been detected in deep vein thrombosis, atherothrombosis, stroke, disseminated intravascular coagulation and trauma. We have previously shown that haemodynamic forces trigger rapid NETosis within sterile occlusive thrombi in vitro. Here, we tested the effects of thrombin, fibrin and fibrinolysis on shear-induced NETosis by imaging NETs with Sytox Green during microfluidic perfusion of factor XIIa-inhibited or thrombin-inhibited human whole blood over fibrillar collagen (±tissue factor). For perfusions under venous pressure drops (19 mm Hg/mm-clot), thrombin generation did not alter the near-zero level of NET generation. In contrast, production of thrombin/fibrin led to a twofold reduction in neutrophil accumulation and a sixfold reduction in NET generation after 30 minutes of arterial perfusion (163 mm Hg/mm-clot). Exogenously added tissue type plasminogen activator (tPA) drove robust fibrinolysis; however, tPA did not trigger NETosis under venous flow. In contrast, tPA did enhance NET generation in clots subjected to arterial pressure drops. After 45 minutes of arterial perfusion, clots treated with 30 nM tPA had a threefold increase in total NET production and a twofold increase in normalized NET generation (measured as deoxyribonucleic acid:neutrophil) compared with fibrin-rich clots. Blocking fibrin polymerization resulted in similar level of NET release seen in tPA-treated clots, whereas ε-aminocaproic acid abolished the NET-enhancing effect of tPA. Therefore, fibrin suppresses NET generation and the absence of fibrin promotes NETs. We demonstrated that shear-induced NETosis was strongly inversely correlated with fibrin in sterile occlusive clots., Competing Interests: None declared., (Georg Thieme Verlag KG Stuttgart · New York.)

The efficacy of reduced order modeling for transstenotic pressure drop in the coronary arteries is presented. Coronary artery disease is a leading cause of death worldwide and the computation of pressure drop in the coronary arteries has become a standard for evaluating the functional significance of a coronary stenosis. Comprehensive models typically employ three-dimensional (3D) computational fluid dynamics (CFD) to simulate coronary blood flow in order to compute transstenotic pressure drop at the arterial stenosis. In this study, we evaluate the capability of different hydrodynamic models to compute transstenotic pressure drop. Models range from algebraic formulae to one-dimensional (1D), two-dimensional (2D), and 3D time-dependent CFD simulations. Although several algebraic pressure-drop formulae have been proposed in the literature, these models were found to exhibit wide variation in predictions. Nonetheless, we demonstrate an algebraic formula that provides consistent predictions with 3D CFD results for various changes in stenosis severity, morphology, location, and flow rate. The accounting of viscous dissipation and flow separation were found to be significant contributions to accurate reduce order modeling of transstenotic coronary hemodynamics., (Copyright © 2019 by ASME.)

Background: Trauma-induced coagulopathy occurs in about 25% of injured patients and accounts for about 10% of deaths worldwide. Upon injury, hemostatic function may decline due to vascular dysfunction, clotting factor deficiencies, hyperfibrinolysis, and/or platelet dysfunction. We investigated agonist-induced calcium signaling in platelets obtained over time from trauma patients., Methods: Platelets from trauma patients and healthy donors were monitored via intracellular calcium mobilization and flow cytometry markers (α2bβ3 activation, P-selectin display, and phosphatidylserine exposure) following stimulation with a panel of agonists (adenosine 5'-diphosphate sodium salt, U46619, convulxin, PAR-1/4 activating peptides, iloprost) used in isolation or in pairwise tests. Furthermore, healthy donor platelets were tested in heterologous plasma isolated from healthy subjects and trauma patients., Results: When exposed to agonists over the first 24 hours postinjury, trauma patient platelets mobilized less calcium in comparison to healthy platelets. Partial recovery of platelet activity was observed in about a third of patients after 120 hours, although not fully obtaining healthy baseline function. Flow cytometry markers of trauma platelets were similar to healthy platelets prior to stimulation, but were depressed in trauma platelets stimulated with adenosine 5'-diphosphate sodium salt or convulxin. Also, washed healthy platelets showed a significant reduction in calcium mobilization when reconstituted in plasma from trauma patients, relative to healthy plasma, at all plasma doses tested., Conclusion: Platelet dysfunction in trauma patients included poor response to multiple agonists relevant to hemostatic function. Furthermore, the inhibitor effect of patient plasma on healthy platelets suggests that soluble plasma species may downregulate endogenous or transfused platelets during trauma.

We analyzed the role of hydrodynamic interactions in a microfluidic channel flow containing a dilute suspension of micron-scale colloidal spheres (0.03%, 0.1%, 0.3% volume fraction) engineered to adhere onto a collector patch on the channel wall at wall shear rates of 9.3-930 s -1 . Particle-wall adhesion was mediated by single-stranded DNA oligomers grafted onto the spheres and the glass channel wall, producing well-defined interactions via DNA strand base pairing. Particle positions in the flow were evolved using Brownian dynamics simulations in which hydrodynamic interactions between moving particles and the channel walls and/or adhered particles were computed off-line using a series of local simulations that explicitly resolve the fluid flow at the particle scale. By systematically varying the nature of hydrodynamic interactions captured in the Brownian dynamics simulations, we find that the interactions between moving and adhered particles represents the single most important physical element in such models. Once captured sufficiently accurately, the resulting models are able to predict coarse variables such as the overall particle coverage evolution, as well as more subtle characteristics, such as the microstructural distribution of the adhered particles., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Heparin proteoglycans (HEP-PGs) carry standard heparin-mediated anticoagulant properties as well as novel antiplatelet functions, a combination that may be significant for targeting multiple pathways in a single therapy. Recent work developing semisynthetic HEP-PG mimetics has shown promising results also in vivo, however flow conditions in vitro that replicate in vivo hemodynamics have not been reported. In this work, we present several assays (platelet calcium mobilization, aggregometry, microfluidic tests at venous and arterial hemodynamics) to characterize specific mechanistic effects of dual antiplatelet and anticoagulant (APAC) constructs as mimetics of HEP-PGs. Three APACs with different conjugation levels of heparin chains (CL10, CL18, HICL) were shown to decrease platelet deposition to collagen surfaces in PPACK-treated whole blood at venous shear rate (200 s -1 ). FXIIa-inhibited whole blood (CTI: corn trypsin inhibitor, 40 μg/mL) perfused over collagen/tissue factor showed reduced both platelet and fibrin deposition when treated with APACs. IC50 values for platelet and fibrin inhibition were calculated for each molecule at venous shear rate. Increasing the shear rate to arterial flows (1000 s -1 ) and using APAC as the sole anticoagulant, resulted in a more potent antiplatelet effect of APAC, suggesting an added effect on von Willebrand Factor (vWF) function. Additionally, APAC caused an inhibition of calcium mobilization specific to thrombin and collagen stimulation and a dose-dependent reduction in collagen-mediated platelet aggregation. Understanding the sensitivity of APAC activity to shear rate, platelet signaling and procoagulant pathways is important for applications in which APAC administration may have beneficial therapeutic effects., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Microfluidic thrombosis assays allow the control of anticoagulation, hemodynamics, pharmacology, and procoagulant surfaces containing collagen ± tissue factor (TF). With corn trypsin inhibitor (CTI) ranging from low (1-4 μg/mL) to high levels (40-60 μg/mL), the function of Factor XIIa (FXIIa) can be modulated in the presence of low or high surface TF. With high CTI and no collagen/TF in the assay, no thrombin is generated during 15-min microfluidic perfusion. At low CTI (no TF), the generation of FXIa leads to fibrin polymerization at ~300 s after the initiation of flow over collagen, an onset time shortened at zero CTI and prolonged at high CTI. The engagement of FXIa was difficult to observe for clotting on high TF surfaces due to the dominance of the extrinsic pathway. Low TF surfaces allowed observable crosstalk between extrinsic pathway generation of thrombin and thrombin-mediated activation of FXIa, a feedback detected at >5 min and attenuated with polyphosphate inhibitor. From thrombin-antithrombin immunoassay of the effluent of blood flowing over collagen/TF, the majority of thrombin was found captured on intrathrombus fibrin. Additionally, extreme shear rates (>10,000 s -1 ) can generate massive von Willebrand Factor fibers that capture FXIIa and FXIa to drive fibrin generation, an event that facilitates VWF fiber dissolution under fibrinolytic conditions. Finally, we found that occlusive sterile thrombi subjected to pressure drops >70 mm-Hg/mm-clots have interstitial stresses sufficient to drive NETosis. These microfluidic studies highlight the interaction of contact pathway factors with the extrinsic pathway, platelet polyphosphate, VWF fibers, and potentially shear-induced NETs.

Objective: We investigated the coregulation of thrombin and fibrin as blood flows over a procoagulant surface., Approach and Results: Using microfluidic perfusion of factor XIIa-inhibited human whole blood (200 s -1 wall shear rate) over a 250-μm long patch of collagen/TF (tissue factor; ≈1 molecule per μm 2 ) and immunoassays of the effluent for F1.2 (prothrombin fragment 1.2), TAT (thrombin-antithrombin complex), and D-dimer (post-end point plasmin digest), we sought to establish the transient mass balance for clotting under venous flow. F1.2 (but almost no free thrombin detected via TAT assay) continually eluted from clots when fibrin was allowed to form. Low-dose fluorescein-Phe-Pro-Arg-chloromethylketone stained fibrin-bound thrombin-a staining ablated by anti-γ'-fibrinogen or the fibrin inhibitor glypro-arg-pro but highly resistant to 7-minute buffer rinse, demonstrating tight binding of thrombin to γ'-fibrin. With fibrin polymerizing for 500 seconds, 92 000 thrombin molecules and 203 000 clot-associated fibrin monomer equivalents were generated per TF molecule (or per μm 2 ). Fibrin reached 15 mg/mL in the pore space (porosity ≈0.5) of a 15-μm-thick thrombus core by 500 seconds and 30 mg/mL by 800 seconds. For a known rate of ≈60 FPA (fibrinopeptide-A) per thrombin per second, each thrombin molecule generated only 3 fibrin monomer equivalents during 500 seconds, indicating an intraclot thrombin half-life of ≈70 ms, much shorter than its diffusional escape time (≈10 seconds). By 800 seconds, gly-pro-arg-pro allowed 4-fold more F1.2 generation, consistent with gly-pro-arg-pro ablating fibrin's antithrombin-I activity and facilitating thrombin-mediated FXIa activation., Conclusions: Under flow, fibrinogen continually penetrates the clot, and γ'-fibrin regulates thrombin., (© 2018 American Heart Association, Inc.)

Trauma with hypovolemic shock is an extreme pathological state that challenges the body to maintain blood pressure and oxygenation in the face of hemorrhagic blood loss. In conjunction with surgical actions and transfusion therapy, survival requires the patient's blood to maintain hemostasis to stop bleeding. The physics of the problem are multiscale: (a) the systemic circulation sets the global blood pressure in response to blood loss and resuscitation therapy, (b) local tissue perfusion is altered by localized vasoregulatory mechanisms and bleeding, and (c) altered blood and vessel biology resulting from the trauma as well as local hemodynamics control the assembly of clotting components at the site of injury. Building upon ongoing modeling efforts to simulate arterial or venous thrombosis in a diseased vasculature, computer simulation of trauma-induced coagulopathy is an emerging approach to understand patient risk and predict response. Despite uncertainties in quantifying the patient's dynamic injury burden, multiscale systems biology may help link blood biochemistry at the molecular level to multiorgan responses in the bleeding patient. As an important goal of systems modeling, establishing early metrics of a patient's high-dimensional trajectory may help guide transfusion therapy or warn of subsequent later stage bleeding or thrombotic risks. This article is categorized under: Analytical and Computational Methods > Computational Methods Biological Mechanisms > Regulatory Biology Models of Systems Properties and Processes > Mechanistic Models., (© 2018 Wiley Periodicals, Inc.)

Extreme flows can exist within pathological vessel geometries or mechanical assist devices which create complex forces and lead to thrombogenic problems associated with disease. Turbulence and boundary layer separation are difficult to obtain in microfluidics due to the low Reynolds number flow in small channels. However, elongational flows, extreme shear rates and stresses, and stagnation point flows are possible using microfluidics and small perfusion volumes. In this review, a series of microfluidic devices used to study pathological blood flows are described. In an extreme stenosis channel pre-coated with fibrillar collagen that rapidly narrows from 500  μ m to 15  μ m, the plasma von Willebrand Factor (VWF) will elongate and assemble into thick fiber bundles on the collagen. Using a micropost-impingement device, plasma flow impinging on the micropost generates strong elongational and wall shear stresses that trigger the growth of a VWF bundle around the post (no collagen required). Using a stagnation-point device to mimic the zone near flow reattachment, blood can be directly impinged upon a procoagulant surface of collagen and the tissue factor. Clots formed at the stagnation point of flow impingement have a classic core-shell architecture where the core is highly activated (P-selectin positive platelets and fibrin rich). Finally, within occlusive clots that fill a microchannel, the Darcy flow driven by ΔP/L > 70 mm-Hg/mm-clot is sufficient to drive NETosis of entrapped neutrophils, an event not requiring either thrombin or fibrin. Novel microfluidic devices are powerful tools to access physical environments that exist in human disease.

Essentials Platelet packing density in a hemostatic plug limits molecular movement to diffusion. A diffusion-dependent steep thrombin gradient forms radiating outwards from the injury site. Clot retraction affects the steepness of the gradient by increasing platelet packing density. Together, these effects promote hemostatic plug core formation and inhibit unnecessary growth., Summary: Background Hemostasis studies performed in vivo have shown that hemostatic plugs formed after penetrating injuries are characterized by a core of highly activated, densely packed platelets near the injury site, covered by a shell of less activated and loosely packed platelets. Thrombin production occurs near the injury site, further activating platelets and starting the process of platelet mass retraction. Tightening of interplatelet gaps may then prevent the escape and exchange of solutes. Objectives To reconstruct the hemostatic plug macro- and micro-architecture and examine how platelet mass contraction regulates solute transport and solute concentration in the gaps between platelets. Methods Our approach consisted of three parts. First, platelet aggregates formed in vitro under flow were analyzed using scanning electron microscopy to extract data on porosity and gap size distribution. Second, a three-dimensional (3-D) model was constructed with features matching the platelet aggregates formed in vitro. Finally, the 3-D model was integrated with volume and morphology measurements of hemostatic plugs formed in vivo to determine how solutes move within the platelet plug microenvironment. Results The results show that the hemostatic mass is characterized by extremely narrow gaps, porosity values even smaller than previously estimated and stagnant plasma velocity. Importantly, the concentration of a chemical species released within the platelet mass increases as the gaps between platelets shrink. Conclusions Platelet mass retraction provides a physical mechanism to establish steep chemical concentration gradients that determine the extent of platelet activation and account for the core-and-shell architecture observed in vivo., (© 2018 International Society on Thrombosis and Haemostasis.)

The study of viscous fluid flow coupled with rigid or deformable solids has many applications in biological and engineering problems, e.g., blood cell transport, drug delivery, and particulate flow. We developed a partitioned approach to solve this coupled Multiphysics problem. The fluid motion was solved by Palabos (Parallel Lattice Boltzmann Solver), while the solid displacement and deformation was simulated by LAMMPS (Large-scale Atomic/Molecular Massively Parallel Simulator). The coupling was achieved through the immersed boundary method (IBM). The code modeled both rigid and deformable solids exposed to flow. The code was validated with the Jeffery orbits of an ellipsoid particle in shear flow, red blood cell stretching test, and effective blood viscosity flowing in tubes. It demonstrated essentially linear scaling from 512 to 8192 cores for both strong and weak scaling cases. The computing time for the coupling increased with the solid fraction. An example of the fluid-solid coupling was given for flexible filaments (drug carriers) transport in a flowing blood cell suspensions, highlighting the advantages and capabilities of the developed code.

The structure and growth of a blood clot depend on the localization of tissue factor (TF), which can trigger clotting during the hemostatic process or promote thrombosis when exposed to blood under pathological conditions. We sought to understand how the growth, structure, and mechanical properties of clots under flow are shaped by the simultaneously varying TF surface density and its exposure area. We used an eight-channel microfluidic device equipped with a 20- or 100-μm-long collagen surface patterned with lipidated TF of surface densities ∼0.1 and ∼2 molecules/μm 2 . Human whole blood was perfused at venous shear, and clot growth was continually measured. Using our recently developed computational model of clot formation, we performed simulations to gain insights into the clot's structure and its resistance to blood flow. An increase in TF exposure area resulted not only in accelerated bulk platelet, thrombin, and fibrin accumulation, but also in increased height of the platelet mass and increased clot resistance to flow. Moreover, increasing the TF surface density or exposure area enhanced platelet deposition by approximately twofold, and thrombin and fibrin generation by greater than threefold, thereby increasing both clot size and its viscous resistance. Finally, TF effects on blood flow occlusion were more pronounced for the longer thrombogenic surface than for the shorter one. Our results suggest that TF surface density and its exposure area can independently enhance both the clot's occlusivity and its resistance to blood flow. These findings provide, to our knowledge, new insights into how TF affects thrombus growth in time and space under flow., (Copyright © 2018 Biophysical Society. All rights reserved.)

Essentials Neutrophil extracellular traps (NETs) are generated during thrombosis and sepsis. The effect of hemodynamics on NETosis during sterile thrombosis was studied using microfluidics. Pressure gradients > 70 mmHg per mm-clot across sterile occlusions drive shear-induced NETosis. High interstitial hemodynamic forces trigger rapid NET release., Summary: Background Neutrophil extracellular traps (NETs) are released when neutrophils encounter infectious pathogens, especially during sepsis. Additionally, NETosis occurs during venous and arterial thrombosis, disseminated intravascular coagulation, and trauma. Objective To determine whether hemodynamic forces trigger NETosis during sterile thrombosis. Methods NETs were imaged with Sytox Green during microfluidic perfusion of activated factor XII-inhibited or thrombin-inhibited human whole blood over fibrillar collagen (with or without tissue factor). Results For perfusions at initial inlet venous or arterial wall shear rates (100 s -1 or 1000 s -1 ), platelets rapidly accumulated and occluded microchannels with subsequent neutrophil infiltration under either flow condition; however, NETosis was detected only in the arterial condition. The level of shear-induced NETs (SINs) at 30 min was > 150-fold higher in the arterial condition in the absence of thrombin and > 80-fold greater in the presence of thrombin than the level in the venous condition. With or without thrombin, venous perfusion for 15 min generated no NETs, but an abrupt shift-up to arterial perfusion triggered NETosis within 2 min, NETs eventually reaching levels 15 min later that were 60-fold greater than that in microchannels without perfusion shift-up. SINs contained citrullinated histone H3 and myeloperoxidase, and were DNase-sensitive, but were not blocked by inhibitors of platelet-neutrophil adhesion, high-mobility group protein box 1-receptor for advanced glycation end products interaction, cyclooxygenase, ATP/ADP, or peptidylarginine deiminase 4. For measured pressure gradients exceeding 70 mmHg per millimeter of clot across NET-generating occlusions to drive interstitial flow, the calculated fluid shear stress on neutrophils exceeded the known lytic value of 150 dyne cm -2 . Conclusions High interstitial hemodynamic forces can drive physically entrapped neutrophils to rapidly release NETs during sterile occlusive thrombosis., (© 2017 International Society on Thrombosis and Haemostasis.)

During clotting under flow, platelets bind and activate on collagen and release autocrinic factors such as ADP and thromboxane, while tissue factor (TF) on the damaged wall leads to localized thrombin generation. Towards patient-specific simulation of thrombosis, a multiscale approach was developed to account for: platelet signalling [neural network (NN) trained by pairwise agonist scanning (PAS), PAS-NN], platelet positions (lattice kinetic Monte Carlo, LKMC), wall-generated thrombin and platelet-released ADP/thromboxane convection-diffusion (partial differential equation, PDE) and flow over a growing clot (lattice Boltzmann). LKMC included shear-driven platelet aggregate restructuring. The PDEs for thrombin, ADP and thromboxane were solved by finite element method using cell activation-driven adaptive triangular meshing. At all times, intracellular calcium was known for each platelet by PAS-NN in response to its unique exposure to local collagen, ADP, thromboxane and thrombin. When compared with microfluidic experiments of human blood clotting on collagen/TF driven by constant pressure drop, the model accurately predicted clot morphology and growth with time. In experiments and simulations at TF at 0.1 and 10 molecule-TF/$\mu$m$^{2}$ and initial wall shear rate of 200 s$^{-1}$, the occlusive blockade of flow for a 60-$\mu$m channel occurred relatively abruptly at 600 and 400 s, respectively (with no occlusion at zero TF). Prior to occlusion, intrathrombus concentrations reached 50 nM thrombin, ~ 1 $\mu$M thromboxane and ~ 10 $\mu$M ADP, while the wall shear rate on the rough clot peaked at ~ 1000-2000 s$^{-1}$. Additionally, clotting on TF/collagen was accurately simulated for modulators of platelet cyclooxygenase-1, P2Y$&#95;{1}$ and IP-receptor. This multiscale approach facilitates patient-specific simulation of thrombosis under hemodynamic and pharmacological conditions., (© The authors 2016. Published by Oxford University Press on behalf of the Institute of Mathematics and its Applications. All rights reserved.)

Introduction: In regions of flow separation/reattachment within diseased arteries, the local hemodynamics can result in stagnation point flow that provides an atypical environment in atherosclerosis. Impinging flows occur with recirculation eddies distal of coronary stenosis or diseased carotid bifurcations., Methods: By perfusing whole blood directly perpendicular to a fibrillar collagen thrombotic surface, a microfluidic device produced a stagnation point flow. Side view visualization of thrombosis in this assay allowed for observation of clot structure and composition at various flow rates and blood biochemistry conditions., Results: For clotting over collagen/tissue factor surfaces, platelet thrombi formed in this device displayed a core-shell architecture with a fibrin-rich, platelet P-selectin-positive core and an outer platelet P-selectin-negative shell. VWF was detected in clots at low and high shear, but when N-acetylcysteine was added to the whole blood, both platelet and VWF deposition were markedly decreased at either low or high flow. To further examine the source of clot stability, 1 mM GPRP was added to prevent fibrin formation while allowing the PAR1/4-cleaving activity of thrombin to progress. The inhibition of fibrin polymerization did not change the overall structure of the clots, demonstrating the stability of these clots without fibrin., Conclusion: Impinging flow microfluidics generate thrombi with a core-shell structure., Competing Interests: Conflicts of Interests B.A. Herbig and S.L. Diamond declare that they have no conflicts of interest.

Essentials Collagen and thrombin when used simultaneously generate highly activated platelets. The effect of thrombin stimulation on subsequent glycoprotein VI (GPVI) function was observed. Soluble fibrin, but not protease activated receptor (PAR) activation, prevented GPVI activation. Circulating soluble fibrin in coagulopathic blood may cause an acquired GPVI signaling defect., Summary: Background In coagulopathic blood, circulating thrombin may drive platelet dysfunction. Methods/Results Using calcium dye-loaded platelets, the effect of thrombin exposure and soluble fibrin generation on subsequent platelet GPVI function was investigated. Exposure of apixaban-treated platelet-rich plasma (12% PRP) to thrombin (1-10 nm), but not ADP or thromboxane mimetic U46619 exposure, dramatically blocked subsequent GPVI activation by convulxin, collagen-related peptide or fibrillar collagen. Consistent with soluble fibrin multimerizing and binding GPVI, the onset of convulxin insensitivity required 200-500 s of thrombin exposure, was not mimicked by exposure to PAR-1/4 activating peptides, was not observed with washed platelets, and was blocked by fibrin polymerization inhibitor (GPRP) or factor XIIIa inhibitor (T101). PAR-1 signaling through Gα q was not required because vorapaxar blocked thrombin-induced calcium mobilization but had no effect on the ability of thrombin to impair GPVI-signaling. Convulxin insensitivity was unaffected by the metalloprotease inhibitor GM6001 or the α II b β 3 antagonist GR144053, indicating negligible roles for GPVI shedding or α II b β 3 binding of fibrin. Thrombin treatment of washed platelets resuspended in purified fibrinogen also produced convulxin insensitivity that was prevented by GPRP. Exposure of apixaban/PPACK-treated whole blood to thrombin-treated fibrinogen resulted in > 50% decrease in platelet deposition in a collagen microfluidic assay that required soluble fibrin assembly. Conclusions Conversion of only 1% plasma fibrinogen in coagulopathic blood would generate 90 nm soluble fibrin, far exceeding ~1 nmGPVI in blood. Soluble fibrin, rather than thrombin-induced platelet activation throuh PAR-1 and PAR-4, downregulated GPVI-signaling in response to stimuli, and may lead to subsequent hypofunction of endogenous or transfused platelets., (© 2017 International Society on Thrombosis and Haemostasis.)

Over the last decade, the coagulation factor XIIa (FXIIa) has seen renewed interest as a therapeutic target. Indeed, its inhibition could offer a protection against thrombosis without increasing the risk of bleeding. Moreover, it could answer the need for a safe prevention of blood-contacting medical devices-related thrombosis. Among the FXII and FXIIa inhibitors already described in literature, organic small-molecular-weight inhibitors are rather left behind. In this study, we were focused on the discovery and assessment of water soluble small molecules. First, a search within our library of compounds flagged two promising hits. Indeed, enzymes and plasma assays suggested they have a greater activity on the contact factors (FXIa, plasma kallikrein and FXIIa) than on the TF pathway. Then, simple pharmacomodulations were undertaken with the aim to design more selective FXIIa inhibitors. This afforded compounds having different degrees of selectivity. All compounds were finally screened in whole blood using an 8-channel microfluidic model and thromboelastometry measurements. Interestingly, all molecules interfered with the thrombus formation and one of them could be considered as a small organic contact inhibitor., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Introduction: Factor VIII (FVIII) or factor IX (FIX)-deficient haemophilic patients display deficits in platelet and fibrin deposition under flow detectable in microfluidics. Compared to fibrin generation, decreased platelet deposition in haemophilic blood flow is more easily rescued with recombinant factor VIIa (rFVIIa), whereas rFVIIa requires FXIIa participation to generate fibrin when tissue factor (TF) is absent., Aims: Perfusion of haemophilic whole blood (WB) over collagen/TF surfaces was used to determine whether rFVIIa/TF was sufficient to bypass poor FIXa/FVIIIa function in blood from patients with haemophilia A and B., Methods: Whole blood treated with high-dose corn trypsin inhibitor (40 μg mL -1 ) from seven healthy donors and 10 patients was perfused over fibrillar collagen presenting low or high TF (TF low or TF high ) at wall shear rate of 100 s -1 ., Results: With WB from healthy controls, platelet deposition and fibrin accumulation increased as TF increased. Factor-deficient WB (1-3% of normal) displayed striking deficits in platelet deposition and fibrin formation at either TF low or TF high . In contrast, mildly factor-deficient WB (14-32%) supported fibrin formation under flow on TF high /collagen. With either TF low or TF high , exogenously added rFVIIa (20 nm) increased platelet deposition and fibrin accumulation in WB from factor-deficient patients (1-3% of normal) to levels commensurate with untreated healthy WB., Conclusion: The absence of FIXa/FVIIIa in patients with severe haemophilia results in deficits in fibrin formation that cannot be rescued by wall-derived TF ex vivo. The effects of rFVIIa on platelet adhesion and rFVIIa/TF can act together to reinforce thrombin generation, platelet deposition and fibrin formation under flow., (© 2017 John Wiley & Sons Ltd.)

Background: Platelet cross-linking during arterial thrombosis involves von Willebrand Factor (VWF) multimers. Therefore, proteolysis of VWF appears promising to disaggregate platelet-rich thrombi and restore vessel patency in acute thrombotic disorders such as ischemic stroke, acute coronary syndrome, or acute limb ischemia. N -Acetylcysteine (NAC, a clinically approved mucolytic drug) can reduce intrachain disulfide bonds in large polymeric proteins. In the present study, we postulated that NAC might cleave the VWF multimers inside occlusive thrombi, thereby leading to their dissolution and arterial recanalization., Methods: Experimental models of thrombotic stroke induced by either intra-arterial thrombin injection or ferric chloride application followed by measurement of cerebral blood flow using a combination of laser Doppler flowmetry and MRI were performed to uncover the effects of NAC on arterial thrombi. To investigate the effect of NAC on larger vessels, we also performed ferric chloride-induced carotid artery thrombosis. In vitro experiments were performed to study the molecular bases of NAC thrombolytic effect, including platelet aggregometry, platelet-rich thrombi lysis assays, thromboelastography (ROTEM), and high-shear VWF string formation using microfluidic devices. We also investigated the putative prohemorrhagic effect of NAC in a mouse model of intracranial hemorrhage induced by in situ collagenase type VII injection., Results: We demonstrated that intravenous NAC administration promotes lysis of arterial thrombi that are resistant to conventional approaches such as recombinant tissue-type plasminogen activator, direct thrombin inhibitors, and antiplatelet treatments. Through in vitro and in vivo experiments, we provide evidence that the molecular target underlying the thrombolytic effects of NAC is principally the VWF that cross-link platelets in arterial thrombi. Coadministration of NAC and a nonpeptidic GpIIb/IIIa inhibitor further improved its thrombolytic efficacy, essentially by accelerating thrombus dissolution and preventing rethrombosis. Thus, in a new large-vessel thromboembolic stroke model in mice, this cotreatment significantly improved ischemic lesion size and neurological outcome. It is important to note that NAC did not worsen hemorrhagic stroke outcome, suggesting that it exerts thrombolytic effects without significantly impairing normal hemostasis., Conclusions: We provide evidence that NAC is an effective and safe alternative to currently available antithrombotic agents to restore vessel patency after arterial occlusion., (© 2017 American Heart Association, Inc.)

Shunt thrombosis remains a major cause of morbidity and mortality, especially during the initial palliation for single-ventricle physiology. The authors present evidence that the P2Y 12 inhibitor cangrelor may fill a therapeutic void in thromboprophylaxis. They base this theory on results showing that platelets from neonatal patients with cyanotic congenital heart disease have a robust response to adenosine diphosphate and are amenable to P2Y 12 inhibition with cangrelor. Unique to this study was their ability to establish drug efficacy in an avatar mouse model that permits the in vivo evaluation of human platelet-mediated thrombus formation illustrating that this P2Y 12 inhibitor yields the intended biological response.