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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.

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.)

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.

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.)

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 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.)

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$_{1}$ 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.)

Platelets present a number of intracellular and transmembrane targets subject to pharmacological modulation, either for cardiovascular disease reduction or as an unintended drug response. Microfluidic devices allow human blood to clot on a defined surface under controlled hemodynamic and pharmacological conditions. The potencies of a number of antiplatelet and anticancer drugs have been tested with respect to platelet deposition on collagen under flow. Inhibitors of cyclooxygenase-1 (COX-1) reduce platelet deposition, either when added ex vivo to blood or ingested orally by patients prior to testing. Some individuals display a functional "aspirin-insensitivity" in microfluidic assay. When certain nonsteroidal anti-inflammatory drugs (NSAIDs) are taken orally, they block COX-1 acetylation by aspirin with concomitant reduction of aspirin efficacy against platelets in microfluidic assay. Both P2Y 1 and P2Y 12 inhibitors reduce platelet deposition under flow, as do NO donors and iloprost that target the guanylate cyclase and the prostacyclin receptor, respectively. In a microfluidic assay of 37 kinase inhibitors, dasatinib had potent antiplatelet activity, while bosutinib was less potent. Dasatinib and bosutinib have known profiles against numerous kinases, revealing overlapping and nonoverlapping activities relevant to their unique actions against platelets. Also, dasatinib caused a marked and specific inhibition of GPVI signaling induced by convulxin, consistent with a dasatinib-associated bleeding risk. Microfluidic devices facilitate drug library screening, dose-response testing, and drug-drug interaction studies. Kinase inhibitors developed as anticancer agents may present antiplatelet activities that are detectable by microfluidic assay and potentially linked to bleeding risks.

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., (© 2016 International Society on Thrombosis and Haemostasis.)

Hemostasis occurs in two different topological scenarios: complete severing of a vessel or disruption of the vessel wall. Either to meet the daily rigors of active life or during an acute trauma, hemostasis involves the regulated and self-limiting production of thrombin to stop bleeding. In contrast, arterial and venous thrombosis typically involves the unregulated, intraluminal growth of a clot, in the absence of bleeding. For either hemostasis or thrombosis, the presence of flow and pressure gradients (delta-P, ΔP) dictates when and where thrombin and fibrin are located and in what quantity. For hemostatic clots, fibrin formation helped limit clot growth. We found that γ'-fibrinogen had a role in limiting clot growth via anti-thrombin activity at venous, but not arterial conditions. For hemophilic blood, severe factor deficiency (<1% healthy) led to a defect in both platelet and fibrin deposition under flow. However, moderate deficiency, which is associated with a less severe bleeding phenotype, had normalized platelet function but still lacked fibrin production. We conclude signaling levels of thrombin can be generated during moderate hemophilia to sufficiently activate platelets to achieve primary hemostasis, even if fibrin formation remains defective. Finally, as a clot grows, shear stresses can become sufficiently extreme in diseased arteries to drive von Willebrand Factor self-association into massive fibers, potentially the final burst of clot growth towards full thrombotic occlusion., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The systems analysis of thrombosis seeks to quantitatively predict blood function in a given vascular wall and hemodynamic context. Relevant to both venous and arterial thrombosis, a Blood Systems Biology approach should provide metrics for rate and molecular mechanisms of clot growth, thrombotic risk, pharmacological response, and utility of new therapeutic targets. As a rapidly created multicellular aggregate with a polymerized fibrin matrix, blood clots result from hundreds of unique reactions within and around platelets propagating in space and time under hemodynamic conditions. Coronary artery thrombosis is dominated by atherosclerotic plaque rupture, complex pulsatile flows through stenotic regions producing high wall shear stresses, and plaque-derived tissue factor driving thrombin production. In contrast, venous thrombosis is dominated by stasis or depressed flows, endothelial inflammation, white blood cell-derived tissue factor, and ample red blood cell incorporation. By imaging vessels, patient-specific assessment using computational fluid dynamics provides an estimate of local hemodynamics and fractional flow reserve. High-dimensional ex vivo phenotyping of platelet and coagulation can now power multiscale computer simulations at the subcellular to cellular to whole vessel scale of heart attacks or strokes. In addition, an integrated systems biology approach can rank safety and efficacy metrics of various pharmacological interventions or clinical trial designs., (© 2016 American Heart Association, Inc.)

Background: Relevant to trauma-induced coagulopathy diagnostics, microfluidic assays allow controlled hemodynamics for testing of platelet and coagulation function using whole blood., Methods: Hemodilution or hyperfibrinolysis was studied under flow with modified healthy whole blood. Furthermore, platelet function was also measured using whole blood from trauma patients admitted to a Level I trauma center. Platelet deposition was measured with PPACK-inhibited blood perfused over collagen surfaces at a wall shear rate of 200 s, whereas platelet/fibrin deposition was measured with corn trypsin inhibitor-treated blood perfused over tissue factor (TF)/collagen., Results: In hemodilution studies, PPACK-treated blood displayed almost no platelet deposition when diluted to 10% hematocrit with saline, platelet-poor plasma, or platelet-rich plasma. Using similar dilutions, platelet/fibrin deposition was essentially absent for corn trypsin inhibitor-treated blood perfused over TF/collagen. To mimic hyperfibrinolysis during trauma, exogenous tissue plasminogen activator (50 nM) was added to blood before perfusion over TF/collagen. At both venous and arterial flows, the generation and subsequent lysis of fibrin were detectable within 6 minutes, with lysis blocked by addition of the plasmin inhibitor, ε-aminocaproic acid. Microfluidic assay of PPACK-inhibited whole blood from trauma patients revealed striking defects in collagen response and secondary platelet aggregation in 14 of 21 patients, whereas platelet hyperfunction was detected in three of 20 patients., Conclusion: Rapid microfluidic detection of (1) hemodilution-dependent impairment of clotting, (2) clot instability because of lysis, (3) blockade of fibrinolysis, or (4) platelet dysfunction during trauma may provide novel diagnostic opportunities to predict trauma-induced coagulopathy risk.

Despite their clinical significance, human platelets are not amenable to genetic manipulation, thus forcing a reliance on mouse models. Culture-derived platelets (CDPs) from human peripheral blood CD34(+) cells can be genetically altered and may eventually be used for transfusions. By use of microfluidics, the time-dependent incorporation of CD41(+)CD42(+) CDPs into clots was measured using only 54,000 CDPs doped into 27 μL of human whole blood perfused over collagen at a wall shear rate of 100 sec(-1). With the use of fluorescence-labeled human platelets (instead of CDPs) doped between 0.25% and 2% of total platelets, incorporation was highly quantitative and allowed monitoring of the anti-αIIbβ3 antagonism that occurred after collagen adhesion. CDPs were only 15% as efficient as human platelets in their incorporation into human thrombi under flow, although both cell types were equally antagonized by αIIbβ3 inhibition. Transient transfection allowed the monitoring of GFP(+) human CDP incorporation into clots. This assay quantifies genetically altered CDP function under flow., (Copyright © 2015 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.)

Factor XIIa (FXIIa) and factor XIa (FXIa) contribute to thrombosis in animal models, whereas platelet-derived polyphosphate (polyP) may potentiate contact or thrombin-feedback pathways. The significance of these mediators in human blood under thrombotic flow conditions on tissue factor (TF) -bearing surfaces remains inadequately resolved. Human blood (corn trypsin inhibitor treated [4 μg/mL]) was tested by microfluidic assay for clotting on collagen/TF at TF surface concentration ([TF]wall) from ∼0.1 to 2 molecules per μm(2). Anti-FXI antibodies (14E11 and O1A6) or polyP-binding protein (PPXbd) were used to block FXIIa-dependent FXI activation, FXIa-dependent factor IX (FIX) activation, or platelet-derived polyP, respectively. Fibrin formation was sensitive to 14E11 at 0 to 0.1 molecules per µm(2) and sensitive to O1A6 at 0 to 0.2 molecules per µm(2). However, neither antibody reduced fibrin generation at ∼2 molecules per µm(2) when the extrinsic pathway became dominant. Interestingly, PPXbd reduced fibrin generation at low [TF]wall (0.1 molecules per µm(2)) but not at zero or high [TF]wall, suggesting a role for polyP distinct from FXIIa activation and requiring low extrinsic pathway participation. Regardless of [TF]wall, PPXbd enhanced fibrin sensitivity to tissue plasminogen activator and promoted clot retraction during fibrinolysis concomitant with an observed PPXbd-mediated reduction of fibrin fiber diameter. This is the first detection of endogenous polyP function in human blood under thrombotic flow conditions. When triggered by low [TF]wall, thrombosis may be druggable by contact pathway inhibition, although thrombolytic susceptibility may benefit from polyP antagonism regardless of [TF]wall., (© 2015 by The American Society of Hematology.)

In prior microfluidic studies with haemophilic blood perfused over collagen, we found that a severe deficiency (<1% factor level) reduced platelet and fibrin deposition, while a moderate deficiency (1-5%) only reduced fibrin deposition. We investigated: (i) the differential effect of rFVIIa (0.04-20 nm) on platelet and fibrin deposition, and (ii) the contribution of the contact pathway to rFVIIa-induced haemophilic blood clotting. Haemophilic or healthy blood with low and high corn trypsin inhibitor (CTI, 4 or 40 μg mL(-1) ) was perfused over collagen at an initial venous wall shear rate of 100 s(-1) . At 100 s(-1) wall shear rate, where FXIIa leads to thrombin production without added tissue factor, FXI-deficient blood (3%) or severely FVIII-deficient blood (<1%) produced no fibrin at either CTI level. Whereas rFVIIa potently enhanced platelet deposition, fibrin generation was not rescued. Distinct from the high CTI condition, engagement of the contact pathway (low CTI) in moderately FVIII-deficient (3%) or moderately FIX-deficient blood (5%) resulted in enhanced platelet and fibrin deposition following 4 nm rFVIIa supplementation. In mildly FVIII-deficient blood (15%) at <24 h since haemostatic therapy, rFVIIa enhanced both platelet and fibrin generation in either CTI condition although fibrin was produced more quickly and abundantly in low CTI. For tissue factor-free conditions of severe haemophilic blood clotting, we conclude that rFVIIa reliably generates low levels of 'signaling' thrombin sufficient to enhance platelet deposition on collagen, but is insufficient to drive fibrin polymerization unless potentiated by the contact pathway., (© 2014 John Wiley & Sons Ltd.)

Since platelet intracellular calcium mobilization [Ca(t)]i controls granule release, cyclooxygenase-1 and integrin activation, and phosphatidylserine exposure, blood clotting simulations require prediction of platelet [Ca(t)]i in response to combinatorial agonists. Pairwise Agonist Scanning (PAS) deployed all single and pairwise combinations of six agonists (ADP, convulxin, thrombin, U46619, iloprost and GSNO used at 0.1, 1, and 10xEC50; 154 conditions including a null condition) to stimulate platelet P2Y1/P2Y12 GPVI, PAR1/PAR4, TP, IP receptors, and guanylate cyclase, respectively, in Factor Xa-inhibited (250 nM apixaban), diluted platelet rich plasma that had been loaded with the calcium dye Fluo-4 NW. PAS of 10 healthy donors provided [Ca(t)]i data for training 10 neural networks (NN, 2-layer/12-nodes) per donor. Trinary stimulations were then conducted at all 0.1x and 1xEC50 doses (160 conditions) as was a sampling of 45 higher ordered combinations (four to six agonists). The NN-ensemble average was a calcium calculator that accurately predicted [Ca (t)]i beyond the single and binary training set for trinary stimulations (R = 0.924). The 160 trinary synergy scores, a normalized metric of signaling crosstalk, were also well predicted (R = 0.850) as were the calcium dynamics (R = 0.871) and high-dimensional synergy scores (R = 0.695) for the 45 higher ordered conditions. The calculator even predicted sequential addition experiments (n = 54 conditions, R = 0.921). NN-ensemble is a fast calcium calculator, ideal for multiscale clotting simulations that include spatiotemporal concentrations of ADP, collagen, thrombin, thromboxane, prostacyclin, and nitric oxide.

The cardiovascular safety of nonsteroidal antiinflammatory drugs (NSAIDs) may be influenced by interactions with antiplatelet doses of aspirin. We sought to quantitate precisely the propensity of commonly consumed NSAIDs—ibuprofen, naproxen, and celecoxib—to cause a drug-drug interaction with aspirin in vivo by measuring the target engagement of aspirin directly by MS. We developed a novel assay of cyclooxygenase-1 (COX-1) acetylation in platelets isolated from volunteers who were administered aspirin and used conventional and microfluidic assays to evaluate platelet function. Although ibuprofen, naproxen, and celecoxib all had the potential to compete with the access of aspirin to the substrate binding channel of COX-1 in vitro, exposure of volunteers to a single therapeutic dose of each NSAID followed by 325 mg aspirin revealed a potent drug-drug interaction between ibuprofen and aspirin and between naproxen and aspirin but not between celecoxib and aspirin. The imprecision of estimates of aspirin consumption and the differential impact on the ability of aspirin to inactivate platelet COX-1 will confound head-to-head comparisons of distinct NSAIDs in ongoing clinical studies designed to measure their cardiovascular risk.

A high-fidelity computational model using a 3D immersed boundary method is used to study platelet dynamics in whole blood. We focus on the 3D effects of the platelet-red blood cell (RBC) interaction on platelet margination and near-wall dynamics in a shear flow. We find that the RBC distribution in whole blood becomes naturally anisotropic and creates local clusters and cavities. A platelet can enter a cavity and use it as an express lane for a fast margination toward the wall. Once near the wall, the 3D nature of the platelet-RBC interaction results in a significant platelet movement in the transverse (vorticity) direction and leads to anisotropic platelet diffusion within the RBC-depleted zone or cell-free layer (CFL). We find that the anisotropy in platelet motion further leads to the formation of platelet clusters, even in the absence of any platelet-platelet adhesion. The transverse motion, and the size and number of the platelet clusters are observed to increase with decreasing CFL thickness. The 3D nature of the platelet-RBC collision also induces fluctuations in off-shear plane orientation and, hence, a rotational diffusion of the platelets. Although most marginated platelets are observed to tumble just outside the RBC-rich zone, platelets further inside the CFL are observed to flow with an intermittent dynamics that alters between sliding and tumbling, as a result of the off-shear plane rotational diffusion, bringing them even closer to the wall. To our knowledge, these new findings are based on the fundamentally 3D nature of the platelet-RBC interaction, and they underscore the importance of using cellular-scale 3D models of whole blood to understand platelet margination and near-wall platelet dynamics., (Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Resting platelets maintain a stable level of low cytoplasmic calcium ([Ca(2+)]cyt) and high dense tubular system calcium ([Ca(2+)]dts). During thrombosis, activators cause a transient rise in inositol trisphosphate (IP3) to trigger calcium mobilization from stores and elevation of [Ca(2+)]cyt. Another major source of [Ca(2+)]cyt elevation is store-operated calcium entry (SOCE) through plasmalemmal calcium channels that open in response to store depletion as [Ca(2+)]dts drops. A 34-species systems model employed kinetics describing IP3-receptor, DTS-plasmalemma puncta formation, SOCE via assembly of STIM1 and Orai1, and the plasmalemma and sarco/endoplasmic reticulum Ca(2+)-ATPases. Four constraints were imposed: calcium homeostasis before activation; stable in zero extracellular calcium; IP3-activatable; and functional SOCE. Using a Monte Carlo method to sample three unknown parameters and nine initial concentrations in a 12-dimensional space near measured or expected values, we found that model configurations that were responsive to stimuli and demonstrated significant SOCE required high inner membrane electric potential (>-70 mV) and low resting IP3 concentrations. The absence of puncta in resting cells was required to prevent spontaneous store depletion in calcium-free media. Ten-fold increases in IP3 caused saturated calcium mobilization. This systems model represents a critical step in being able to predict platelets' phenotypes during hemostasis or thrombosis., (Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Background: Coagulation factor deficiencies create a range of bleeding phenotypes. Microfluidic devices offer controlled hemodynamics and defined procoagulant triggers for measurement of clotting under flow., Objectives: We tested a flow assay of contact pathway-triggered clotting to quantify platelet and fibrin deposition distal of dysfunctional thrombin production. Microfluidic metrics were then compared with PTT or % factor activity assays., Methods: Whole blood (WB) treated with low level corn trypsin inhibitor (4 μg mL⁻¹) from nine healthy donors and 27 patients (deficient in factor [F] VIII, 19 patients; FIX, one patient; FXI, one patient; VWF, six patients) was perfused over fibrillar collagen at wall shear rate = 100 s⁻¹., Results: Using healthy WB, platelets deposited within 30 s, while fibrin appeared within 6 min. Compared with healthy controls, WB from patients displayed a 50% reduction in platelet deposition only at < 1% factor activity. In contrast, striking defects in fibrin deposition occurred for patients with < 13% factor activity (or PTT > 40 s). Full occlusion of the 60-μm high channel was completely absent over the 15-min test in patients with < 1% factor activity, while an intermediate defect was present in patients with > 1% factor., Conclusion: Spontaneous bleeding in patients with < 1% factor activity may be linked to deficits in both platelet and fibrin deposition, a risk known to be mitigated when factor levels are raised to > 1% activity (PTT of ~40-60 s), a level that does not necessarily rescue fibrin formation under flow., (© 2013 International Society on Thrombosis and Haemostasis.)

Background: Microfluidic devices recreate the hemodynamic conditions of thrombosis., Methods: Whole blood inhibited with PPACK was treated ex vivo with inhibitors and perfused over collagen for 300 s (wall shear rate=200 s(-1)) using a microfluidic flow assay. Platelet accumulation was measured in the presence of COX-1 inhibitor (aspirin, ASA), P2Y₁ inhibitor (MRS 2179), P2Y₁₂ inhibitor (2MeSAMP) or combined P2Y1 and P2Y₁₂ inhibitors., Results: High dose ASA (500 μM), 2MeSAMP (100 μM), MRS 2179 (10 μM), or combined 2MeSAMP and MRS 2179 decreased total platelet accumulation by 27.5%, 75.6%, 77.7%, and 87.9% (p<0.01), respectively. ASA reduced secondary aggregation rate between 150 and 300 s without effect on primary deposition rate on collagen from 60 to 150 s. In contrast, 2MeSAMP and MRS 2179 acted earlier and reduced primary deposition to collagen between 60 and 105 s and secondary aggregation between 105 and 300 s. R(COX) and R(P2Y) (defined as a ratio of secondary aggregation rate to primary deposition rate) demonstrated 9 of 10 subjects had R(COX)<1 or R(P2Y)<1 following ASA or 2MeSAMP addition, while 6 of 10 subjects had R(P2Y)<1 following MRS 2179 addition. Combined MRS 2179 and 2MeSAMP inhibited primary platelet deposition rate and platelet secondary aggregation beyond that of each individual inhibitor. Receiver-Operator Characteristic area under the curve (AUC) indicated the robustness of R(COX) and R(P2Y) to detect inhibition of secondary platelet aggregation by ASA, 2MeSAMP, and MRS 2179 (AUC of 0.874 0.966, and 0.889, respectively)., Conclusions: Microfluidic devices can detect platelet sensitivity to antiplatelet agents. The R-value can serve as a self-normalized metric of platelet function for a single blood sample., (© 2013.)

Background: Microfluidic devices can create hemodynamic conditions for platelet assays. We validated an 8-channel device in a study of interdonor response to acetylsalicylic acid (ASA, aspirin) with whole blood from 28 healthy individuals., Methods: Platelet deposition was assessed before treatment or 24 h after ingestion of 325 mg ASA. Whole blood (plus 100 μmol/L H-d-Phe-Pro-Arg-chloromethylketone to inhibit thrombin) was further treated ex vivo with ASA (0-500 μmol/L) and perfused over fibrillar collagen for 300 s at a venous wall shear rate (200 s(-1))., Results: Ex vivo ASA addition to blood drawn before aspirin ingestion caused a reduction in platelet deposition [half-maximal inhibitory concentration (IC50) approximately 10-20 μmol/L], especially between 150 and 300 s of perfusion, when secondary aggregation mediated by thromboxane was expected. Twenty-seven of 28 individuals displayed smaller deposits (45% mean reduction; range 10%-90%; P < 0.001) from blood obtained 24 h after ASA ingestion (no ASA added ex vivo). In replicate tests, an R value to score secondary aggregation [deposition rate from 150 to 300 s normalized by rate from 60 to 150 s] showed R < 1 in only 2 of 28 individuals without ASA ingestion, with R > 1 in only 3 of 28 individuals after 500 μmol/L ASA addition ex vivo. At 24 h after ASA ingestion, 21 of 28 individuals displayed poor secondary aggregation (R < 1) without ex vivo ASA addition, whereas the 7 individuals with residual secondary aggregation (R > 1) displayed insensitivity to ex vivo ASA addition. Platelet deposition was not correlated with platelet count. Ex vivo ASA addition caused similar inhibition at venous and arterial wall shear rates., Conclusions: Microfluidic devices quantified platelet deposition after ingestion or ex vivo addition of aspirin.

We present three-dimensional numerical simulations of hydrodynamic interaction between a red blood cell (RBC) and a platelet in a wall-bounded shear flow. The dynamics and large deformation of the RBC are fully resolved in the simulations using a front-tracking method. The objective is to quantify the influence of tank treading and tumbling dynamics of the RBC, and the presence of a bounding wall on the deflection of platelet trajectories. We observe two types of interaction: A crossing event in which the platelet comes in close proximity to the RBC, rolls over it, and continues to move in the same direction; and a turning event in which the platelet turns away before coming close to the RBC. The crossing events occur when the initial lateral separation between the cells is above a critical separation, and the turning events occur when it is below the critical separation. The critical lateral separation is found to be higher during the tumbling motion than that during the tank treading. When the RBC is flowing closer to the wall than the platelet, the critical separation increases by several fold, implying the turning events have higher probability to occur than the crossing events. On the contrary, if the platelet is flowing closer to the wall than the RBC, the critical separation decreases by several folds, implying the crossing events are likely to occur. Based on the numerical results, we propose a mechanism of continual platelet drift from the RBC-rich region of the vessel towards the wall by a succession of turning and crossing events. The trajectory deflection in the crossing events is found to depend nonmonotonically on the initial lateral separation, unlike the monotonic trend observed in tracer particle deflection and in deformable sphere-sphere collision. This nonmonotonic trend is shown to be a consequence of the deformation of the RBC caused by the platelet upon collision. An estimation of the platelet diffusion coefficient yields values that are similar to those reported in experiments and computer simulations with multicellular suspension.

Understanding the response of cells to multiple stimuli is vital for predicting donor specific responses and better understanding the signaling pathways involved. This is of particular importance in platelets because exposure of phosphatidylserine (PS) occurs upon costimulation but not with a single agonist. Here, we describe a multiplexed pairwise agonist scanning-flow cytometry (PAS-FC) method of measuring platelet inside-out responses to all pairs of six platelet agonists (convulxin, SFLLRN, AYPGKF, ADP, U46619, and PGE(2)) used at their EC(50) concentrations. These agonists allowed exploration of platelet signaling downstream of GPVI, PAR-1, PAR-4, P2Y(1), P2Y(12), TP, and IP receptors. The three-color flow cytometry method simultaneously measured integrin α(IIb)β(3) activation with PAC-1 antibody, P-selectin exposure (via α granule release) with anti-P-selectin, and PS exposure with annexin V. These responses were consistent across a healthy male donor pool. In duplicate measurements with each donor, 4 of the 10 donors had a sufficiently unique 45-parameter (15 pairs × 3 colors) phenotype to self-cluster (P < 0.001). This method has the potential for efficiently scanning for patient specific responses across a broad agonist-receptor space.

Achieving hemostasis following vascular injury requires the rapid accumulation of platelets and fibrin. Here we used a combination of confocal intravital imaging, genetically engineered mice, and antiplatelet agents to determine how variations in the extent of platelet activation following vascular injury arise from the integration of different elements of the platelet-signaling network. Two forms of penetrating injury were used to evoke the hemostatic response. Both produced a hierarchically organized structure in which a core of fully activated platelets was overlaid with an unstable shell of less-activated platelets. This structure emerged as hemostasis was achieved and persisted for at least 60 minutes following injury, its organization at least partly reflecting agonist concentration gradients. Thrombin activity and fibrin formation were found primarily in the innermost core. As proposed previously, greater packing density in the core facilitated contact-dependent signaling and limited entry of plasma-borne molecules visualized with fluorophores coupled to dextran and albumin. Blocking contact-dependent signaling or inhibiting thrombin reduced the size of the core, while the shell was heavily influenced by adenosine 5'-diphosphate and regulators of Gi2-mediated signaling. Thus, the hemostatic response is shown to produce a hierarchical structure arising, in part, from distinct elements of the platelet-signaling network.

Objective: In severe stenosis, von Willebrand factor (vWF) experiences millisecond exposures to pathological wall shear rates (γ(w)). We sought to evaluate the deposition of vWF onto collagen surfaces under flow in these environments., Methods and Results: Distinct from viscometry experiments that last many seconds, we deployed microfluidic devices for single-pass perfusion of whole blood or platelet-free plasma over fibrillar type 1 collagen (<50 ms transit time) at pathological γ(w) or spatial wall shear rate gradients (grad γ(w)). Using fluorescent anti-vWF, long thick vWF fibers (>20 μm) bound to collagen were visualized at constant γ(w)>30000 s(-1) during perfusion of platelet-free plasma, a process enhanced by EDTA. Rapid acceleration or deceleration of EDTA platelet-free plasma at grad γ(w)=±1.1×10(5) to ±4.3×10(7) s(-1)/cm did not promote vWF deposition. At 19400 s(-1), EDTA blood perfusion resulted in rolling vWF-platelet nets, although blood perfusion (normal Ca(2+)) generated large vWF/platelet deposits that repeatedly embolized and were blocked by anti-glycoprotein Ib or the α(IIb)β(3) inhibitor GR144053 and did not require grad γ(w). Blood perfusion at venous shear rate (200 s(-1)) produced a stable platelet deposit that was a substrate for massive but unstable vWF-platelet aggregates when flow was increased to 7800 s(-1)., Conclusions: Triggered by collagen and enhanced by platelet glycoprotein Ib and α(IIb)β(3), vWF fiber formation occurred during acute exposures to pathological γ(w) and did not require gradients in wall shear rate.

Background: Thrombin undergoes convective and diffusive transport, making it difficult to visualize during thrombosis. We developed the first sensor capable of revealing inner clot thrombin dynamics., Methods and Results: An N-terminal-azido thrombin-sensitive fluorescent peptide (ThS-P) with a thrombin-releasable quencher was linked to anti-CD41 using click chemistry to generate a thrombin-sensitive platelet binding sensor (ThS-Ab). Rapid thrombin cleavage of ThS-P (K(m) = 40.3 μm, k(cat) = 1.5 s(-1) ) allowed thrombin monitoring by ThS-P or ThS-Ab in blood treated with 2-25 pm tissue factor (TF). Individual platelets had > 20-fold more ThS-Ab fluorescence after clotting. In a microfluidic assay of whole blood perfusion over collagen ± linked TF (wall shear rate = 100 s(-1) ), ThS-Ab fluorescence increased between 90 and 450 s for 0.1-1 molecule-TF μm(-2) and co-localized with platelets near fibrin. Without TF, neither thrombin nor fibrin was detected on the platelet deposits by 450 s. Using a microfluidic device to control the pressure drop across a thrombus forming on a porous collagen/TF plug (521 s(-1) ), thrombin and fibrin were detected at the thrombus-collagen interface at a zero pressure drop, whereas 80% less thrombin was detected at 3200 Pa in concert with fibrin polymerizing within the collagen. With anti-mouse CD41 ThS-Ab deployed in a mouse laser injury model, the highest levels of thrombin arose between 40 and 160 s nearest the injury site where fibrin co-localized and where the thrombus was most mechanically stable., Conclusion: ThS-Ab reveals thrombin locality, which depends on surface TF, flow and intrathrombus pressure gradients., (© 2012 International Society on Thrombosis and Haemostasis.)

During thrombotic or hemostatic episodes, platelets bind collagen and release ADP and thromboxane A(2), recruiting additional platelets to a growing deposit that distorts the flow field. Prediction of clotting function under hemodynamic conditions for a patient's platelet phenotype remains a challenge. A platelet signaling phenotype was obtained for 3 healthy donors using pairwise agonist scanning, in which calcium dye-loaded platelets were exposed to pairwise combinations of ADP, U46619, and convulxin to activate the P2Y(1)/P2Y(12), TP, and GPVI receptors, respectively, with and without the prostacyclin receptor agonist iloprost. A neural network model was trained on each donor's pairwise agonist scanning experiment and then embedded into a multiscale Monte Carlo simulation of donor-specific platelet deposition under flow. The simulations were compared directly with microfluidic experiments of whole blood flowing over collagen at 200 and 1000/s wall shear rate. The simulations predicted the ranked order of drug sensitivity for indomethacin, aspirin, MRS-2179 (a P2Y(1) inhibitor), and iloprost. Consistent with measurement and simulation, one donor displayed larger clots and another presented with indomethacin resistance (revealing a novel heterozygote TP-V241G mutation). In silico representations of a subject's platelet phenotype allowed prediction of blood function under flow, essential for identifying patient-specific risks, drug responses, and novel genotypes.

Objective: At sites of vascular injury, thrombin is an important mediator in thrombus growth and stability. Using microfluidic flow devices as well as patterned surfaces of collagen and tissue factor (TF), we sought to determine the role that fibrin plays in clot stability without interfering with the production of thrombin., Methods and Results: We deployed an 8-channel microfluidic device to study coagulation during corn trypsin inhibitor-treated (XIIa-inhibited) whole blood perfusion over lipidated TF linked to a fibrillar collagen type 1 surface. Clot growth and embolization were measured at initial inlet venous (200 s(-1)) or arterial (1000 s(-1)) wall shear rates under constant flow rate or pressure relief mode in the presence or absence of Gly-Pro-Arg-Pro (GPRP) to block fibrin polymerization. Numerical calculations for each mode defined hemodynamic forces on the growing thrombi. In either mode at inlet venous flow, increasing amounts of TF on the surface led to a modest dose-dependent increase (up to 2-fold) in platelet deposition, but resulted in massive fibrin accumulation (>50-fold) only when exceeding a critical TF threshold. At a venous inlet flow, GPRP led to a slight 20% increase in platelet accumulation (P<0.01) in pressure relief mode with thrombi resisting ≈1500 s(-1) before full channel occlusion. GPRP-treated thrombi were unstable under constant flow rate, where shear forces caused embolization at a maximum shear rate of ≈2300 s(-1) (69 dynes/cm2). In constant flow rate mode, the nonocclusive platelet-fibrin deposits (no GPRP) withstood maximum shear rates of ≈29 000 s(-1) (870 dyne/cm2) at ≈95% of full channel occlusion. For arterial inlet shear rate, embolization was marked for either mode with GPRP present when shear forces reached 87 dynes/cm2 (≈2900 s(-1)). Under constant flow rate, platelet-fibrin deposits (no GPRP) withstood maximums of 2400 dynes/cm2 (80,000 s(-1)) at ≈90% of full channel occlusion prior to embolization., Conclusions: Fibrin increased clot strength by 12- to 28-fold. Under pressure relief mode, ≈2-fold more fibrin was produced under venous flow (P<0.001). These studies define embolization criteria for clots formed with surface TF-triggered thrombin production (±fibrin) under venous and arterial flows.

Microfluidic devices allow for the controlled perfusion of human or mouse blood over defined prothrombotic surfaces at venous and arterial shear rates. To mimic in vivo injuries such a plaque rupture, the need exists to link lipidated tissue factor (TF) to surface-bound collagen fibers. Recombinant TF was relipidated in liposomes of phosphatidylserine/phosphatidylcholine/biotin-linked phosphatidylethanolamine (20:79:1 PS/PC/bPE molar ratio). Collagen was patterned in a 250-μm-wide stripe and labeled with biotinylated anticollagen antibody which was then bound with streptavidin, allowing the subsequent capture of the TF liposomes. To verify and detect the TF liposome-collagen assembly, individual molecular complexes of TF-factor VIIa on collagen were visualized using the proximity ligation assay (PLA) to produce discretely localized fluorescent events that were strictly dependent on the presence of factor VIIa and primary antibodies against TF or factor VIIa. Perfusion for 450 s (wall shear rate, 200 s(-1)) of corn trypsin inhibitor (CTI, a factor XIIa inhibitor) treated whole blood over the stripe of TF-collagen enhanced platelet adhesion by 30 ± 8% (p < 0.001) and produced measurable fibrin (>50-fold increase) as compared to surfaces lacking TF. PS/PC/bPE liposomes lacking TF resulted in no enhancement of platelet deposition. Essentially no fibrin was formed during perfusion over collagen surfaces or collagen surfaces with liposomes lacking TF despite the robust platelet deposition, indicating a lack of kinetically significant platelet-borne tissue factor in healthy donor blood. This study demonstrates a reliable approach to link functionally active TF to collagen for microfluidic thrombosis studies.

In a focal injury model, platelets adhere and activate under flow on a collagen-coated surface, creating a field of individual platelet aggregates. These aggregates exhibit distinct structural characteristics that are linked to the local flow conditions. By combining image analysis techniques and epifluorescence microscopy, we developed a robust strategy for quantifying the characteristic instantaneous width and length of a growing platelet deposit. We have confirmed the technique using model images consisting of ellipsoid objects and quantified the shear rate-dependent nature of aggregate morphology. Venous wall shear rate conditions (100 s(-1)) generated small, circular platelet deposits, whereas elevated arterial shear rates (500 and 1000 s(-1)) generated platelet masses elongated twofold in the direction of flow. At 2000 s(-1), an important regime for von Willebrand Factor (vWF)-mediated recruitment, we observed sporadic platelet capture events on collagen that led to rapidly growing deposits. Furthermore, inter-donor differences were investigated with respect to aggregate growth rate. After perfusion at elevated shear rates (1000 s(-1)) for 5 min, we identified a twofold increase in aggregate size (81.5 ± 24.6 μm; p < 0.1) and a threefold increase in growth rate parallel to the flow (0.40 ± 0.09 μm/s; p < 0.01) for an individual donor. Suspecting a role for vWF, we found that this donor had a twofold increase in soluble vWF relative to the other donors and pooled plasma. Microfluidic devices in combination with automated morphology analysis offer new tools for characterizing clot development under flow.

Although much is known about extrinsic regulators of platelet function such as nitric oxide and prostaglandin I(2) (PGI(2)), considerably less is known about intrinsic mechanisms that prevent overly robust platelet activation after vascular injury. Here we provide the first evidence that regulators of G-protein signaling (RGS) proteins serve this role in platelets, using mice with a G184S substitution in G(i2α) that blocks RGS/G(i2) interactions to examine the consequences of lifting constraints on G(i2)-dependent signaling without altering receptor:effector coupling. The results show that the G(i2α)(G184S) allele enhances platelet aggregation in vitro and increases platelet accumulation after vascular injury when expressed either as a global knock-in or limited to hematopoietic cells. Biochemical studies show that these changes occur in concert with an attenuated rise in cyclic adenosine monophosphate levels in response to prostacyclin and a substantial increase in basal Akt activation. In contrast, basal cyclic adenosine monophosphate (cAMP) levels, agonist-stimulated increases in [Ca(++)](i), Rap1 activation, and α-granule secretion were unaffected. Collectively, these observations (1) demonstrate an active role for RGS proteins in regulating platelet responsiveness, (2) show that this occurs in a pathway-selective manner, and (3) suggest that RGS proteins help to prevent unwarranted platelet activation as well as limiting the magnitude of the normal hemostatic response.

Prediction of cellular response to multiple stimuli is central to evaluating patient-specific clinical status and to basic understanding of cell biology. Cross-talk between signaling pathways cannot be predicted by studying them in isolation and the combinatorial complexity of multiple agonists acting together prohibits an exhaustive exploration of the complete experimental space. Here we describe pairwise agonist scanning (PAS), a strategy that trains a neural network model based on measurements of cellular responses to individual and all pairwise combinations of input signals. We apply PAS to predict calcium signaling responses of human platelets in EDTA-treated plasma to six different agonists (ADP, convulxin, U46619, SFLLRN, AYPGKF and PGE(2)) at three concentrations (0.1, 1 and 10 x EC(50)). The model predicted responses to sequentially added agonists, to ternary combinations of agonists and to 45 different combinations of four to six agonists (R = 0.88). Furthermore, we use PAS to distinguish between the phenotypic responses of platelets from ten donors. Training neural networks with pairs of stimuli across the dose-response regime represents an efficient approach for predicting complex signal integration in a patient-specific disease milieu.

To quantify how various molecular mechanisms are integrated to maintain platelet homeostasis and allow responsiveness to adenosine diphosphate (ADP), we developed a computational model of the human platelet. Existing kinetic information for 77 reactions, 132 fixed kinetic rate constants, and 70 species was combined with electrochemical calculations, measurements of platelet ultrastructure, novel experimental results, and published single-cell data. The model accurately predicted: (1) steady-state resting concentrations for intracellular calcium, inositol 1,4,5-trisphosphate, diacylglycerol, phosphatidic acid, phosphatidylinositol, phosphatidylinositol phosphate, and phosphatidylinositol 4,5-bisphosphate; (2) transient increases in intracellular calcium, inositol 1,4,5-trisphosphate, and G(q)-GTP in response to ADP; and (3) the volume of the platelet dense tubular system. A more stringent test of the model involved stochastic simulation of individual platelets, which display an asynchronous calcium spiking behavior in response to ADP. Simulations accurately reproduced the broad frequency distribution of measured spiking events and demonstrated that asynchronous spiking was a consequence of stochastic fluctuations resulting from the small volume of the platelet. The model also provided insights into possible mechanisms of negative-feedback signaling, the relative potency of platelet agonists, and cell-to-cell variation across platelet populations. This integrative approach to platelet biology offers a novel and complementary strategy to traditional reductionist methods.

The flux of platelet agonists into flowing blood is a critical event in thrombosis and hemostasis. However, few in vitro methods exist for examining and controlling the role of platelet agonists on clot formation and stability under hemodynamic conditions. In this paper, we describe a membrane-based method for introducing a solute into flowing blood at a defined flux. The device consisted of a track-etched polycarbonate membrane reversibly sealed between two microfluidic channels; one channel contained blood flowing at a physiologically relevant shear rate, and the other channel contained the agonist(s). An analytical model described the solute flux as a function of the membrane permeability and transmembrane pressure. The model was validated using luciferase as a model solute for transmembrane pressures of 50-400 Pa. As a proof-of-concept, the weak platelet agonist ADP was introduced into whole blood flowing at 250 s(-1) at three fluxes (1.5, 2.4, and 4.4 x 10(-18) mol microm(-2) s(-1)). Platelet aggregation was monitored by fluorescence microscopy during the experiment and the morphology of aggregates was determined by post hoc confocal and electron microscopy. At the lowest flux (1.5 x 10(-18) mol microm(-2) s(-1)), we observed little to no aggregation. At the higher fluxes, we observed monolayer (2.4 x 10(-18) mol microm(-2) s(-1)) and multilayer (4.4 x 10(-18) mol microm(-2) s(-1)) aggregates of platelets and found that the platelet density within an aggregate increased with increasing ADP flux. We expect this device to be a useful tool in unraveling the role of platelet agonists on clot formation and stability.

Human neutrophil proteases cathepsin G and elastase can directly alter platelet function and/or participate in coagulation cascade reactions on the platelet or neutrophil surface to enhance fibrin formation. The clotting of recalcified platelet-free plasma (PFP) or platelet-rich plasma (PRP) supplemented with corn trypsin inhibitor (to shut down contact activation) was studied in well-plates or flow assays. Inhibitors of cathepsin G or elastase significantly delayed the burst time (t(50)) of thrombin generation in neutrophil-supplemented PRP from 49 min to 59 and 77 min, respectively, in well-plate assays as well as reduced neutrophil-promoted fibrin deposition on fibrinogen-adherent platelets under flow conditions. In flow assays, purified cathepsin G was a far more potent activator of platelet-dependent coagulation than elastase. Anti-tissue factor had no effect on neutrophil protease-enhanced thrombin formation in PRP. The addition of cathepsin G (425 nm) or convulxin (10 nm) to PRP dramatically reduced the t(50) of thrombin generation from 53 min to 17 or 23 min, respectively. In contrast, the addition of elastase to PRP left the t(50) unaltered. Whereas perfusion of PFP (gamma(w) = 62.5 s(-1)) over fibrinogen-adherent platelets did not result in fibrin formation until 50 min, massive fibrin could be observed on cathepsin G-treated platelets even at 35 min. Cathepsin G addition to corn trypsin inhibitor-treated PFP produced little thrombin unless anionic phospholipid was present. However, further activation inhibition studies indicated that cathepsin G enhances fibrin deposition under flow conditions by elevating the activation state of fibrinogen-adherent platelets rather than by cleaving coagulation factors.

We examined the effect of adherent neutrophils on fibrin deposition under laminar flow conditions. Perfusion of recalcified citrated platelet-free plasma (PFP) over neutrophils adherent to fibrinogen-coated glass at a venous wall shear rate of 62.5 s(-1) for 15 minutes resulted in dense deposition of fibrin around each neutrophil, whereas fibrin deposition on glass alone was sparse. Fibrin deposition on neutrophils was markedly reduced by anti-CD18 or anti-CD11b or a higher shear rate (250 s(-1)). Significantly less fibrin was deposited around adherent fibrinogen-coated beads, indicating that nonspecific "cross-sectional capture" effects were not responsible for the massive fibrin deposition on neutrophils. Direct visualization of fibrin capture by neutrophils and elimination of fibrin deposition at 15 minutes by a factor XIIa inhibitor (50 microg/mL corn trypsin inhibitor [CTI]) or elastase/cathepsin G inhibitors (Methoxysuccinyl-Ala-Ala-Pro-Ala-Chloromethyl-Ketone/Z-Gly-Leu-Phe-CMK, 100 micromol/L) indicated that neutrophils can capture short fibrin strands flowing in recalcified PFP lacking CTI and can also promote thrombin generation through pathways attenuated by inhibitors of factor XIIa, elastase, and cathepsin G. When neutrophils were allowed to interact with platelets on a fibrinogen surface before perfusion of recalcified CTI-treated PFP, the fibrin deposition was observed to be dramatic compared with that over surfaces coated with platelets alone or neutrophils alone and compared with that formed on platelets adherent to collagen. This neutrophil promotion of platelet-mediated fibrin formation was attenuated by inhibitors of elastase or cathepsin G but not anti-tissue factor antibody. Neutrophils can interact with platelets via released proteases to increase platelet procoagulant activity and fibrin formation in CTI-treated plasma under the low-flow conditions expected in venous thrombosis or inflammation.

Adhesion and subsequent aggregation between neutrophils and platelets is dependent upon the initial binding of P-selectin on activated platelets to P-selectin glycoprotein ligand 1 (PSGL-1) on the microvilli of neutrophils. High speed, high resolution videomicroscopy of flowing neutrophils interacting with spread platelets demonstrated that thin membrane tethers were pulled from neutrophils in 32 +/- 4% of the interactions. After capture by spread platelets, neutrophil membrane tethers (length of 5.9 +/- 4.1 microm, n = 63) were pulled at an average rate of 6-40 microm/s as the wall shear rate was increased from 100-250 s(-1). The average tether lifetime decreased significantly (P < 0.001) from 630 to 133 ms as the shear rate was increased from 100 s(-1) (F(bond) = 86 pN) to 250 s(-1) (F(bond) = 172 pN), which is consistent with P-selectin/PSGL-1 bond dynamics under stress. Tether formation was blocked by antibodies against P-selectin or PSGL-1, but not by anti-CD18 antibodies. During neutrophil rolling on P-selectin at 150 s(-1), thin membrane tethers were also pulled from the neutrophils. The characteristic jerking motion of the neutrophil coexisted with tether growth (8.9 +/- 8.8 microm long), whereas tether breakage (average lifetime of 3.79 +/- 3.32 s) caused an acute jump in the rolling velocity, proving multiple bonding in the cell surface and the tether surface contact area. Extremely long membrane tethers (>40 microm) were sometimes pulled, which detached in a flow-dependent mechanism of microparticle formation. Membrane tethers were also formed when neutrophils were perfused over platelet monolayers. These results are the first visualization of the often hypothesized tethers that shield the P-selectin/PSGL-1 bond from force loading to regulate neutrophil rolling during inflammation and thrombosis.

The heterotypic aggregation of cell mixtures or colloidal particles such as proteins occurs in a variety of settings such as thrombosis, immunology, cell separations, and diagnostics. Using the set of population balance equations (PBEs) to predict dynamic aggregate size and composition distributions is not feasible. The stochastic algorithm of Gillespie for chemical reactions (. J. Comput. Phys. 22:403-434) was reformulated to simulate the kinetic behavior of aggregating systems. The resulting Monte Carlo (MC) algorithm permits exact calculation of the decay rates of monomers and the temporally evolving distribution of sizes and compositions of the aggregates. Moreover, it permits calculation of all moments of these distributions. Using this method, we explored the heterotypic aggregation of fully activated platelets and neutrophils in a linear shear flow of shear rate G = 335 s(-1). At plasma concentrations, the half-lives of homotypically aggregating platelet and neutrophil singlets were 8.5 and 2.4 s, respectively. However, for heterotypic aggregation, the half-lives for platelets and neutrophils decreased to 2.0 and 0.11 s, respectively, demonstrating that flowing neutrophils accelerate capture of platelets and growth of aggregates. The required number of calculations per time step of the MC algorithm was typically a small fraction of Omega(1/2), where Omega is the initial number of particles in the system, making this the fastest MC method available. The speed of the algorithm makes feasible the deconvolution of kernels for general biological heterotypic aggregation processes.

We have modeled platelet aggregation in a linear shear flow by accounting for two body collision hydrodynamics, platelet activation and receptor biology. Considering platelets and their aggregates as unequal-sized spheres with DLVO interactions (psi(platelet) = -15 mV, Hamaker constant = 10(-19) J), detailed hydrodynamics provided the flow field around the colliding platelets. Trajectory calculations were performed to obtain the far upstream cross-sectional area and the particle flux through this area provided the collision frequency. Only a fraction of platelets brought together by a shearing fluid flow were held together if successfully bound by fibrinogen cross-bridging GPIIb/IIIa receptors on the platelet surfaces. This fraction was calculated by modeling receptor-mediated aggregation using the formalism of Bell (Bell, G. I. 1979. A theoretical model for adhesion between cells mediated by multivalent ligands. Cell Biophys. 1:133-147) where the forward rate of bond formation dictated aggregation during collision and was estimated from the diffusional limited rate of lateral association of receptors multiplied by an effectiveness factor, eta, to give an apparent rate. For a value of eta = 0.0178, we calculated the overall efficiency (including both receptor binding and hydrodynamics effects) for equal-sized platelets with 50,000 receptors/platelet to be 0.206 for G = 41.9 s(-1), 0.05 for G = 335 s(-1), and 0.0086 for G = 1920 s(-1), values which are in agreement with efficiencies determined from initial platelet singlet consumption rates in flow through a tube. From our analysis, we predict that bond formation proceeds at a rate of approximately 0.1925 bonds/microm2 per ms, which is approximately 50-fold slower than the diffusion limited rate of association. This value of eta is also consistent with a colloidal stability of unactivated platelets at low shear rates. Fibrinogen was calculated to mediate aggregation quite efficiently at low shear rates but not at high shear rates. Although secondary collisions (an orbitlike trajectory) form only a small fraction of the total number of collisions, they become important at high shear rates (>750 s(-1)), as these are the only collisions that provide enough time to result in successful aggregate formation mediated by fibrinogen. The overall method provides a hydrodynamic and receptor correction of the Smoluchowski collision kernel and gives a first estimate of eta for the fibrinogen-GPIIb/IIIa cross-bridging of platelets. We also predict that secondary collisions extend the shear rate range at which fibrinogen can mediate successful aggregation.