Modeling the heterogeneous apoptotic response of caspase-mediated signaling in tumor cells.

Resisting apoptosis is a hallmark of cancer. For this reason, it may be possible to force cancer cells to die by targeting components along the apoptotic signaling pathway. However, apoptosis signaling is challenging to understand due to dynamic and complex behaviors of ligands, receptors, and intracellular signaling components in response to cancer therapy. In this work, we forecast the apoptotic response based on the combined impact of these features. We expanded a previously established mathematical model of caspase-mediated apoptosis to include extracellular activation and receptor dynamics. In addition, three potential threshold values of caspase-3 necessary for the activation of apoptosis were selected to forecast which cells become apoptotic over time. We first vary ligand and receptor levels with the number of intracellular signaling proteins remaining consistent. Then, we vary the intracellular protein molecules in each simulated tumor cell to forecast the response of a heterogeneous population. By leveraging the benefits of computational modeling, we investigate the combined effect of several factors on the onset of apoptosis. This work provides quantitative insights for how the apoptotic signaling response can be forecasted, and precisely triggered, amongst heterogeneous cells via extracellular activation.

Overcoming the coupled climate and biodiversity crises and their societal impacts.

Earth's biodiversity and human societies face pollution, overconsumption of natural resources, urbanization, demographic shifts, social and economic inequalities, and habitat loss, many of which are exacerbated by climate change. Here, we review links among climate, biodiversity, and society and develop a roadmap toward sustainability. These include limiting warming to 1.5°C and effectively conserving and restoring functional ecosystems on 30 to 50% of land, freshwater, and ocean "scapes." We envision a mosaic of interconnected protected and shared spaces, including intensively used spaces, to strengthen self-sustaining biodiversity, the capacity of people and nature to adapt to and mitigate climate change, and nature's contributions to people. Fostering interlinked human, ecosystem, and planetary health for a livable future urgently requires bold implementation of transformative policy interventions through interconnected institutions, governance, and social systems from local to global levels.

Activation of Most Toll-Like Receptors in Whole Human Blood Attenuates Platelet Deposition on Collagen under Flow.

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 (Pam3CKS4, 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-κB), the IKK-inhibitor (IKK inhibitor VII) and NF-κB inhibitor (Bay 11-7082) were tested. The IKK/NF-κB 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.

Microwave Susceptibility Observation of Interacting Many-Body Andreev States.

Electrostatic charging affects the many-body spectrum of Andreev states, yet its influence on their microwave properties has not been elucidated. We developed a circuit quantum electrodynamics probe that, in addition to transition spectroscopy, measures the microwave susceptibility of different states of a semiconductor nanowire weak link with a single dominant (spin-degenerate) Andreev level. We found that the microwave susceptibility does not exhibit a particle-hole symmetry, which we qualitatively explain as an influence of Coulomb interaction. Moreover, our state-selective measurement reveals a large, π-phase shifted contribution to the response common to all many-body states which can be interpreted as arising from a phase-dependent continuum in the superconducting density of states.

Anti-GPVI Fab reveals distinct roles for GPVI signaling in the first platelet layer and subsequent layers during microfluidic clotting on collagen with or without tissue factor.

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.

Coherent manipulation of an Andreev spin qubit.

Two promising architectures for solid-state quantum information processing are based on electron spins electrostatically confined in semiconductor quantum dots and the collective electrodynamic modes of superconducting circuits. Superconducting electrodynamic qubits involve macroscopic numbers of electrons and offer the advantage of larger coupling, whereas semiconductor spin qubits involve individual electrons trapped in microscopic volumes but are more difficult to link. We combined beneficial aspects of both platforms in the Andreev spin qubit: the spin degree of freedom of an electronic quasiparticle trapped in the supercurrent-carrying Andreev levels of a Josephson semiconductor nanowire. We performed coherent spin manipulation by combining single-shot circuit-quantum-electrodynamics readout and spin-flipping Raman transitions and found a spin-flip time T S = 17 microseconds and a spin coherence time T 2E = 52 nanoseconds. These results herald a regime of supercurrent-mediated coherent spin-photon coupling at the single-quantum level.

Oral health knowledge and practices in the Kaski District of Nepal.

INTRODUCTION: Oral disease is a widespread problem in Nepal. However, up-to-date information on oral health is limited and oral health initiatives may be shaped by assumptions about insufficient oral health knowledge. Furthermore, the influence of socio-demographic factors on oral health in Nepal remains unclear. This study aims to explore the relationship between demographic background and oral health knowledge, attitudes and behaviors in rural Nepal. METHODS: Secondary analysis of data from a community-based survey on oral health knowledge, beliefs, practices, and access to care among residents ages 12 and above across 4 rural villages in Nepal's Kaski District (Total number = 3,243). Chi-square tests were performed to examine associations among oral health knowledge, attitudes and behaviors and demographic characteristics. RESULTS: Participants reported a baseline knowledge of oral health; 92.4% knew about the recommended tooth-brushing regimen. Participants with higher education and younger age demonstrated better oral health knowledge. Misconceptions about dental treatment causing blindness (23.1%), deafness (11.6%), and mental health problems (14.9%) were reported across all groups. CONCLUSION: Numerous factors besides knowledge likely determine individual oral health behavior. Future interventions should consider community-based outreach programs and dental care delivery through community Health Posts to build trust in dental care, build on existing knowledge and community experiences, and improve access to preventative care. Up-to-date understanding of oral health knowledge and practices and sociocultural influences on oral health behavior will better focus interventions and policy decisions.

Thermal shift assay to probe melting of thrombin, fibrinogen, fibrin monomer, and fibrin: Gly-Pro-Arg-Pro induces a fibrin monomer-like state in fibrinogen.

BACKGROUND: Thrombin activates fibrinogen and binds the fibrin E-domain (Kd ~ 2.8 µM) and the splice variant γ'-domain (Kd ~ 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 (Tm) of thrombin, PPACK-thrombin, fibrinogen, fibrin monomer, and fibrin. RESULTS: Large increases in Tm indicated that calcium led to protein stabilization (0 vs. 2 mM Ca2+) 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 Tm of thrombin (58.3 vs. 78.3 °C). Treatment of fibrinogen with fibrin polymerization inhibitor GPRP increased fibrinogen stability by ΔTm = 9.3 °C, similar to the ΔTm when fibrinogen was converted to fibrin monomer (ΔTm = 8.8 °C) or to fibrin (ΔTm = 10.4 °C). Addition of PPACK-thrombin at high 5:1 M ratio to fibrin(ogen) had little effect on fibrin(ogen) Tm 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.

Snap-valve cerebral shunt design for intracranial pressure operation and ultrasound visualization.

Cerebral spinal fluid (CSF) shunts are the main treatment for hydrocephalus. They divert excess CSF from the ventricular system to the abdominal, pleural, or intravascular space where it is absorbed. The shunt valve regulates flow based on intracranial pressure (ICP) to maintain a physiologically stable and safe ICP. Shunt malfunction is difficult to detect, life-threatening and common. The present study demonstrates that snap-though buckling (STB) shells can be transformed into pressure-relief valves that act in the normal physiological range of ICP. Three different shell designs in this preliminary experiment were found to have opening and closing pressures that fall within the physiologically normal range of ICP of 6 to 25 cm H2O. Furthermore, these STB shells demonstrate a valve actuation that is visible by ultrasound and have an implantable form-factor that is similar to currently available shunt valves. The unique characteristics of STB shell valves have potential clinical applications for shunt monitoring using ultrasound imaging and can be fabricated from antibiotic-impregnated materials to mitigate shunt infection. These characteristics make STB valves attractive for future use in cerebral shunt systems.

Hot Nonequilibrium Quasiparticles in Transmon Qubits.

Nonequilibrium quasiparticle excitations degrade the performance of a variety of superconducting circuits. Understanding the energy distribution of these quasiparticles will yield insight into their generation mechanisms, the limitations they impose on superconducting devices, and how to efficiently mitigate quasiparticle-induced qubit decoherence. To probe this energy distribution, we systematically correlate qubit relaxation and excitation with charge-parity switches in an offset-charge-sensitive transmon qubit, and find that quasiparticle-induced excitation events are the dominant mechanism behind the residual excited-state population in our samples. By itself, the observed quasiparticle distribution would limit T_{1} to ≈200 µs, which indicates that quasiparticle loss in our devices is on equal footing with all other loss mechanisms. Furthermore, the measured rate of quasiparticle-induced excitation events is greater than that of relaxation events, which signifies that the quasiparticles are more energetic than would be predicted from a thermal distribution describing their apparent density.

Platelet packing density is an independent regulator of the hemostatic response to injury.

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.

Hemodynamic force triggers rapid NETosis within sterile thrombotic occlusions.

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.

Soluble fibrin causes an acquired platelet glycoprotein VI signaling defect: implications for coagulopathy.

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 Gαq 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 αIIb ß3 antagonist GR144053, indicating negligible roles for GPVI shedding or αIIb ß3 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.

Recombinant factor VIIa addition to haemophilic blood perfused over collagen/tissue factor can sufficiently bypass the factor IXa/VIIIa defect to rescue fibrin generation.

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 (TFlow or TFhigh ) 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 TFlow or TFhigh . In contrast, mildly factor-deficient WB (14-32%) supported fibrin formation under flow on TFhigh /collagen. With either TFlow or TFhigh , 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.

Hierarchical organization of the hemostatic response to penetrating injuries in the mouse macrovasculature.

Essentials Methods were developed to image the hemostatic response in mouse femoral arteries in real time. Penetrating injuries produced thrombi consisting primarily of platelets. Similar to arterioles, a core-shell architecture of platelet activation occurs in the femoral artery. Differences from arterioles included slower platelet activation and reduced thrombin dependence. SUMMARY: Background Intravital studies performed in the mouse microcirculation show that hemostatic thrombi formed after penetrating injuries develop a characteristic architecture in which a core of fully activated, densely packed platelets is overlaid with a shell of less activated platelets. Objective Large differences in hemodynamics and vessel wall biology distinguish arteries from arterioles. Here we asked whether these differences affect the hemostatic response and alter the impact of anticoagulants and antiplatelet agents. Methods Approaches previously developed for intravital imaging in the mouse microcirculation were adapted to the femoral artery, enabling real-time fluorescence imaging despite the markedly thicker vessel wall. Results Arterial thrombi initiated by penetrating injuries developed the core-and-shell architecture previously observed in the microcirculation. However, although platelet accumulation was greater in arterial thrombi, the kinetics of platelet activation were slower. Inhibiting platelet ADP P2Y12 receptors destabilized the shell and reduced thrombus size without affecting the core. Inhibiting thrombin with hirudin suppressed fibrin accumulation, but had little impact on thrombus size. Removing the platelet collagen receptor, glycoprotein VI, had no effect. Conclusions These results (i) demonstrate the feasibility of performing high-speed fluorescence imaging in larger vessels and (ii) highlight differences as well as similarities in the hemostatic response in the macro- and microcirculation. Similarities include the overall core-and-shell architecture. Differences include the slower kinetics of platelet activation and a smaller contribution from thrombin, which may be due in part to the greater thickness of the arterial wall and the correspondingly greater separation of tissue factor from the vessel lumen.

Transport physics and biorheology in the setting of hemostasis and thrombosis.

The biophysics of blood flow can dictate the function of molecules and cells in the vasculature with consequent effects on hemostasis, thrombosis, embolism, and fibrinolysis. Flow and transport dynamics are distinct for (i) hemostasis vs. thrombosis and (ii) venous vs. arterial episodes. Intraclot transport changes dramatically the moment hemostasis is achieved or the moment a thrombus becomes fully occlusive. With platelet concentrations that are 50- to 200-fold greater than platelet-rich plasma, clots formed under flow have a different composition and structure compared with blood clotted statically in a tube. The platelet-rich, core/shell architecture is a prominent feature of self-limiting hemostatic clots formed under flow. Importantly, a critical threshold concentration of surface tissue factor is required for fibrin generation under flow. Once initiated by wall-derived tissue factor, thrombin generation and its spatial propagation within a clot can be modulated by γ'-fibrinogen incorporated into fibrin, engageability of activated factor (FIXa)/activated FVIIIa tenase within the clot, platelet-derived polyphosphate, transclot permeation, and reduction of porosity via platelet retraction. Fibrin imparts tremendous strength to a thrombus to resist embolism up to wall shear stresses of 2400 dyne cm(-2) . Extreme flows, as found in severe vessel stenosis or in mechanical assist devices, can cause von Willebrand factor self-association into massive fibers along with shear-induced platelet activation. Pathological von Willebrand factor fibers are A Disintegrin And Metalloprotease with ThromboSpondin-1 domain 13 resistant but are a substrate for fibrin generation due to FXIIa capture. Recently, microfluidic technologies have enhanced the ability to interrogate blood in the context of stenotic flows, acquired von Willebrand disease, hemophilia, traumatic bleeding, and drug action.

Platelet-targeting thiol reduction sensor detects thiol isomerase activity on activated platelets in mouse and human blood under flow.

UNLABELLED: Essentials Protein disulfide isomerases may have an essential role in thrombus formation. A platelet-binding sensor (PDI-sAb) was developed to detect thiol reductase activity under flow. Primary human platelet adhesion to collagen at 200 s(-1) was correlated with the PDI-sAb signal. Detected thiol reductase activity was localized in the core of growing thrombi at the site of injury in mice. SUMMARY: Background Protein disulfide isomerases (PDIs) may regulate thrombus formation in vivo, although the sources and targets of PDIs are not fully understood. Methods and results Using click chemistry to link anti-CD61 and a C-terminal azido disulfide-linked peptide construct with a quenched reporter, we developed a fluorogenic platelet-targeting antibody (PDI-sAb) for thiol reductase activity detection in whole blood under flow conditions. PDI-sAb was highly responsive to various exogenous reducing agents (dithiothreitol, glutathione and recombinant PDI) and detected thiol reductase activity on P-selectin/phosphatidylserine-positive platelets activated with convulxin/PAR1 agonist peptide, a signal partially blocked by PDI inhibitors and antibody. In a microfluidic thrombosis model using 4 µg mL(-1) corn trypsin inhibitor-treated human blood perfused over collagen (wall shear rate = 100 s(-1) ), the PDI-sAb signal increased mostly over the first 200 s, whereas platelets continually accumulated for over 500 s, indicating that primary adhesion to collagen, but not secondary aggregation, was correlated with the PDI-sAb signal. Rutin and the PDI blocking antibody RL90 reduced platelet adhesion and the PDI-sAb signal only when thrombin production was inhibited with PPACK, suggesting limited effects of platelet thiol isomerase activity on platelet aggregation on collagen in the presence of thrombin. With anti-mouse CD41 PDI-sAb used in an arteriolar laser injury model, thiol reductase activity was localized in the core of growing thrombi where platelets displayed P-selectin and were in close proximity to disrupted endothelium. Conclusion PDI-sAb is a sensitive and real-time reporter of platelet- and vascular-derived disulfide reduction that targets clots as they form under flow to reveal spatial gradients.

Pathological von Willebrand factor fibers resist tissue plasminogen activator and ADAMTS13 while promoting the contact pathway and shear-induced platelet activation.

BACKGROUND: Under severe stenotic conditions, von Willebrand factor (VWF) multimerizes into large insoluble fibers at pathological shear rates. OBJECTIVE: Evaluate the mechanics and biology of VWF fibers without the confounding effects of endothelium or collagen. METHODS: Within a micropost-impingement microfluidic device, > 100-µm long VWF fibers multimerized on the post within 10 min using EDTA-treated platelet-free plasma (PFP) perfused at wall shear rates > 5000 s(-1) . RESULTS: von Willebrand factor fiber thickness increased to > 10 µm as a result of increasing the shear rate to 10,000 s(-1) . In a stress-strain test, fibrous VWF had an elastic modulus of ~50 MPa. The insoluble VWF fibers were non-amyloid because they rapidly dissolved in trypsin, plasmin or 2% SDS, but were resistant to 50 nm ADAMTS13 or 100 nm tissue plasminogen activator in plasma. Following fiber formation, perfusion of low corn trypsin inhibitor (CTI)-treated (4 µg mL(-1) ), recalcified citrated plasma at 1500 s(-1) caused fibrin formation on the VWF fibers, a result not observed with purified type 1 collagen or a naked micropost. During VWF fiber formation, contact pathway factors accumulated on VWF because the use of EDTA/D-Phe-Pro-Arg chloromethylketone (PPACK)/apixaban/high CTI-treated PFP during VWF fiber formation prevented the subsequent fibrin production from low-CTI, recalcified citrated PFP. VWF fibers displayed FXIIa-immunostaining. When PPACK-inhibited whole blood was perfused over VWF fibers, platelets rolled and arrested on the surface of VWF, but only displayed P-selectin if prevailing shear rates were pathological. Platelet arrest on VWF fibers was blocked with αIIb ß3 antagonist GR144053. CONCLUSIONS: We report VWF fiber-contact pathway crosstalk and mechanisms of thrombolytic resistance in hemodynamic settings of myocardial infarction.