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Essentials An optimal therapeutic strategy has yet to be established to prevent early shunt thrombosis. A phase 1 study of cangrelor was performed in neonates after palliation of congenital heart disease. PD endpoint of >90% platelet inhibition in 60% of patients was achieved at 0.5 µg/kg/min dosing. No serious adverse events related to drug administration were observed, including bleeding. ABSTRACT: Background Systemic-to-pulmonary artery shunt thrombosis is a significant cause of early postoperative mortality in neonates after palliation of congenital heart disease. In the context of thromboprophylaxis, an optimal therapeutic strategy has yet to be established before aspirin administration. Cangrelor, a fast-acting, reversible P2Y 12 inhibitor, may fill this unmet need. Objectives To evaluate the pharmacokinetics (PK), pharmacodynamics (PD), and safety of cangrelor in neonates undergoing stage 1 palliation. Methods This prospective, open-label, single-arm study evaluated two cangrelor dosing cohorts following placement of a systemic-to-pulmonary artery shunt, right ventricle-to-pulmonary artery shunt, or ductal stent. Drug concentrations and platelet reactivity, assessed by light transmission aggregometry and in microfluidic assays (MF), were measured. Results Twenty-two patients were consented and 15 received a 1-hour infusion of cangrelor at either 0.5 µg/kg/min (cohort 1) or 0.25 µg/kg/min (cohort 2). Whereas the primary PD endpoint was achieved at the higher dose (ie, reduction in maximal platelet aggregation by ≥90% in 60% of participants), only 29% of those in cohort 2 attained this goal. Comparable and statistically significant results were obtained in MF assays (P < .0001 vs. baseline). Drug levels during infusion were 3-fold higher in cohort 1 vs. cohort 2 (P < .001). Most participants (70%) had undetectable drug levels by 10 minutes postinfusion with full recovery in platelet function at 1 hour. No drug-related bleeding events occurred. Conclusions Favorable PK/PD properties of cangrelor 0.5 µg/kg/min dosing and safety profile warrant further evaluation in neonates following palliative cardiac procedures., (© 2020 International Society on Thrombosis and Haemostasis.)

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.)

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

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

Blood clotting under hemodynamic conditions involves numerous multiscale interactions from the molecular scale to macroscopic vessel and systemic circulation scales. Transmission of shear forces to platelet receptors such as GPIbα, P-selectin, α(2)β(1), and α(2b)β(3) controls adhesion dynamics. These forces also drive membrane tether formation, cellular deformation, and mechanosignaling in blood cells. Blood flow results in red blood cell (RBC) drift towards the center of the vessel along with a near-wall plasma layer enriched with platelets. RBC motions also dramatically enhance platelet dispersion. Trajectories of individual platelets near a thrombotic deposit dictate capture-activation-arrest dynamics as these newly arriving platelets are exposed to chemical gradients of ADP, thromboxane, and thrombin within a micron-scale boundary layer formed around the deposit. If shear forces are sufficiently elevated (>50 dyne/cm(2)), the largest polymers of von Willebrand Factor may elongate with concomitant shear-induced platelet activation. Finally, thrombin generation enhances platelet recruitment and clot strength via fibrin polymerization. By combination of coarse-graining, continuum, and stochastic algorithms, the numerical simulation of the growth rate, composition, and occlusive/embolic potential of a thrombus now spans multiscale phenomena. These simulations accommodate particular flow geometries, blood phenotype, pharmacological regimen, and reactive surfaces to help predict disease risk or response to therapy.

Determination of the patient-specific response to antiplatelet agents facilitates proper dosing for both acute and chronic prophylaxis. "Closed" systems (with or without flow) may fail to predict pharmacological potency in situations where platelets rapidly accumulate under flow conditions at a site of thrombosis ("Open" systems). Using an 8-channel microfluidic flow assay of human whole blood with corn trypsin inhibitor (+/- PPACK) perfused over focal zones of collagen, dose-response curves were measured for pharmacological agents at a wall shear rate of 210 s(-1). The P2Y(1) inhibitor MRS 2179 (IC(50) = 0.233 +/- 0.132 microM) and P2Y(12) inhibitor 2-MeSAMP (IC(50) = 2.558 +/- 0.799 microM) were potent blockers of secondary platelet accumulation under flow, while the P2X(1) inhibitor (NF 449) and apyrase failed to reduce platelet accumulation. MRS 2179 and 2-MeSAMP had undetectable effects on initial platelet adhesion to collagen. Numerical simulation of convective-diffusive transport and apyrase-mediated catalytic degradation of ADP indicated that ultra-high concentrations of apyrase ( approximately 2000 U mL(-1)) would be required to have the same effect under flow as much lower concentrations (1 U mL(-1)) currently used in closed systems (aggregometry or cone-and-plate viscometer). This is the first evaluation of IC(50) values for P2Y(12) and P2Y(1) antagonists under controlled flow conditions. Evaluation of antiplatelet agents in open flow systems demonstrates that inhibition of either ADP by apyrase or antagonism of P2X(1) signaling had no inhibitory effect on platelet accumulation. This technique provides a platform for rapidly investigating effects of antithrombotic therapies simultaneously in a model injury system.

Background: In resting platelets, endothelial cell specific adhesion molecule (ESAM) is located in alpha granules, increasing its cell surface expression following platelet activation. However, the function of ESAM on platelets is unknown., Objective: To determine whether ESAM has a role in thrombus formation., Methods and Results: We found that following platelet activation ESAM localizes to the junctions between adjacent platelets, suggesting a role for this protein in contact-dependent events that regulate thrombus formation. To test this hypothesis we examined the effect of ESAM deletion on platelet function. In vivo, ESAM(-/-) mice achieved more stable hemostasis than wild-type mice following tail transection, and developed larger thrombi following laser injury of cremaster muscle arterioles. In vitro, ESAM(-/-) platelets aggregated at lower concentrations of G protein-dependent agonists than wild-type platelets, and were more resistant to disaggregation. In contrast, agonist-induced calcium mobilization, alpha(IIb)beta(3) activation, alpha-granule secretion and platelet spreading, were normal in ESAM-deficient platelets. To understand the molecular mechanism by which ESAM regulates platelet activity, we utilized a PDZ domain array to identify the scaffold protein NHERF-1 as an ESAM binding protein, and further demonstrated that it associates with ESAM in both resting and activated platelets., Conclusions: These findings support a model in which ESAM localizes to platelet contacts following platelet activation in order to limit thrombus growth and stability so that the optimal hemostatic response occurs following vascular injury.

Background: Flow chambers allow the ex vivo study of platelet response to defined surfaces at controlled wall shear stresses. However, most assays require 1-10 mL of blood and are poorly suited for murine whole blood experiments., Objective: To measure murine platelet deposition and stability in response to focal zones of prothrombotic stimuli using 100 microL of whole blood and controlled flow exposure., Methods: Microfluidic methods were used for patterning acid-soluble collagen in 100 microm x 100 microm patches and creating flow channels with a volume of 150 nL. Within 1 min of collection into PPACK and fluorescent anti-mouse CD41 mAb, whole blood from normal mice or from mice deficient in the integrin alpha(2) subunit was perfused for 5 min over the patterned collagen. Platelet accumulation was measured at venous and arterial wall shear rates. After 5 min, thrombus stability was measured with a 'shear step-up' to 8000 s(-1)., Results: Wild-type murine platelets adhered and aggregated on collagen in a biphasic shear-dependent manner with increased deposition from 100 to 400 s(-1), but decreased deposition at 1000 s(-1). Adhesion to patterned collagen was severely diminished for platelets lacking a functional alpha(2)beta(1) integrin. Those integrin alpha(2)-deficient platelets that did adhere were removed from the surface when challenged to shear step-up. PAR4 agonist (AYPGKF) treatment of the thrombus at 5 min enhanced aggregate stability during the shear step-up., Conclusions: PAR4 signaling enhances aggregate stability by mechanisms independent of other thrombin-dependent pathways such as fibrin formation.

1. The methyl ester prodrug roxifiban is an orally active, potent and selective antagonist of the platelet glycoprotein GPIIb/IIIa receptor and is being developed for the prevention and treatment of arterial thrombosis. 2. Roxifiban was rapidly hydrolyzed to the zwitterion XV459 in vivo and by liver slices from the rat, mouse and human and by intestinal cores from dog. XV459 was metabolized to only a small extent in vitro and in vivo. 3. Studies with rat and dog given radiolabelled roxifiban showed limited oral absorption with the majority of the radiolabel being excreted in faeces. After i.v. doses of 14C-roxifiban, most of the radioactivity was recovered in the urine of rat whereas the dog excreted significant amounts of radioactivity in bile and urine. 4. XV459 could be metabolized extrahepatically by dog gut flora to produce an isoxazoline ring-opened metabolite. In vitro hepatic metabolism of XV459 was mainly by hydroxylation at the prochiral and chiral centres of the isoxazoline ring. These hydroxylated metabolites were not detected in the urine and plasma of human volunteers administered roxifiban. 5. Initial LC/MS identification of metabolites was achieved by dosing the rat with an equimolar mixture of d0:d4 roxifiban and detecting isotopic clusters of pseudomolecular ions. Unequivocal characterization of these metabolites was achieved by LC/MS, LC/NMR and high-field NMR techniques using synthetic standards of the metabolites. 6. The synthesis of one hydroxylated metabolite enabled the assignment of the correct stereochemistry of the substituted hydroxyl group on the isoxazoline ring.

A transport-reaction model describing penetration of plasmin by diffusion and permeation into a dissolving fibrin gel was solved numerically to explore mechanisms that lead to the formation and growth of dissolution fingers through blood clots during thrombolytic therapy. Under conditions of fluid permeation driven by arterial pressures, small random spatial variations in the initial fibrin density within clots (+/-4 to 25% peak variations) were predicted by the simulation to result in dramatic dissolution fingers that grew in time. With in vitro experiments, video microscopy revealed that the shape of the proximal face of a fibrin gel, when deformed by pressure-driven permeation, led to lytic breakthrough in the center of the clot, consistent with model predictions of increased velocities in this region leading to cannulation. Computer simulation of lysis of fibrin retracted by platelets (where more permeable regions are expected in the middle of the clot due to retraction) predicted cannulation of the clot during thrombolysis. This residual, annular thrombus was predicted to lyse more slowly, because radial pressure gradients to drive inner clot permeation were quite small. In conjunction with kinetic models of systemic pharmacodynamics and plasminogen activation biochemistry, a two-dimensional transport-reaction model can facilitate the prediction of the time and causes of clot cannulation, poor reperfusion, and embolism during thrombolysis.

Background: We developed a computer model to predict lysis rates of thrombi for intravenous thrombolytic regimens based on the convective/diffusive penetration of reacting and adsorbing fibrinolytic species from the circulation into the proximal face of a dissolving clot., Methods and Results: Solution of a one-compartment plasma model provided the dynamic concentrations of fibrinolytic species that served as inlet conditions for stimulation of the one-dimensional spatiodynamics within a dissolving fibrin clot of defined composition. The model predicted the circulating levels of tissue plasminogen activator (TPA) and plasminogen levels found in clinical trials for various intravenous therapies. To test the model predictions under in vitro conditions, plasma clots were perfused with TPA (0.1 mumol/L) and plasminogen (1.0 mumol/L) delivered at constant permeation velocity of 0.1 or 0.2 mm/min. The model provided an accurate prediction of the measured lysis front movement. For TPA administration regimens used clinically, simulations predicted clot dissolution rates that were consistent with observed reperfusion times. For unidirectional permeation, the continual accumulation of adsorbing species at the moving lysis front due to prior rounds of solubilization and rebinding was predicted to provide for a marked concentration of TPA and plasmin and the eventual depletion of antiplasmin and macroglobulin in an advancing (approximately 0.25 mm thick) lysis zone., Conclusions: Pressure-driven permeation greatly enhances and is a primary determinant of the overall rate of clot lysis and creates a complex local reaction environment at the plasma/clot interface. With simulation of reaction and transport, it becomes possible to quantitatively link the administration regimen, plasminogena activator properties, and thrombolytic outcome.