• We have developed a biomimetic microfluidic platelet bioreactor that recapitulates bone marrow and blood vessel microenvironments.• Application of shear stress in this bioreactor triggers physiological proplatelet production, and platelet release.Platelet transfusions total >2.17 million apheresis-equivalent units per year in the United States and are derived entirely from human donors, despite clinically significant immunogenicity, associated risk of sepsis, and inventory shortages due to high demand and 5-day shelf life. To take advantage of known physiological drivers of thrombopoiesis, we have developed a microfluidic human platelet bioreactor that recapitulates bone marrow stiffness, extracellular matrix composition, micro-channel size, hemodynamic vascular shear stress, and endothelial cell contacts, and it supports high-resolution live-cell microscopy and quantification of platelet production. Physiological shear stresses triggered proplatelet initiation, reproduced ex vivo bone marrow proplatelet production, and generated functional platelets. Modeling human bone marrow composition and hemodynamics in vitro obviates risks associated with platelet procurement and storage to help meet growing transfusion needs. (Blood. 2014;124(12):1857-1867) IntroductionAlthough platelets (PLTs) play critical roles in hemostasis, 1 angiogenesis, 2 and innate immunity, 3 PLT production remains poorly understood. Consequently, PLT units are derived entirely from human donors, despite serious clinical concerns owing to their immunogenicity and associated risk of sepsis. 4 More than 2.17 million apheresisequivalent PLT units are transfused yearly in the United States 5,6 at a cost of .$1 billion per year. Although demand for PLT transfusions has increased markedly in the past decade, a near-static pool of donors and a 5-day PLT unit shelf life resulting from bacterial contamination 7 and storage-related PLT deterioration, 8 have resulted in significant PLT shortages. 9 Furthermore, artificial platelet substitutes have failed to replace physiological platelet products. 10 An efficient, donorindependent PLT bioreactor capable of generating clinically significant numbers of functional human PLTs is necessary to obviate risks associated with PLT procurement and storage, and help meet growing transfusion needs. In vivo, megakaryocytes (MKs) PLT progenitors sit outside blood vessels in the bone marrow (BM) and extend long, branching cellular structures designated proPLTs into the circulation from which PLTs are released. 11-15 Nearly 100% of human adult MKs must produce ;10 3 PLTs each to account for circulating PLT counts. 16 Although functional human PLTs were first grown in vitro in 1995, 17 to date only ;10% of human MKs initiate proPLT production in culture. This results in yields of 10 122 PLTs per CD34 1 cord blood-derived or embryonic stem cell-derived MK, 18 which are themselves of limited availability, constituting a significant bottleneck in the ex vivo production of a PLT transfusion unit. Although second-generation c...

Intravascular blood clots form in an environment in which hydrodynamic forces dominate and in which fluid-mediated transport is the primary means of moving material. The clotting system has evolved to exploit fluid dynamic mechanisms and to overcome fluid dynamic challenges to ensure that clots that preserve vascular integrity can form over the wide range of flow conditions found in the circulation. Fluid-mediated interactions between the many large deformable red blood cells and the few small rigid platelets lead to high platelet concentrations near vessel walls where platelets contribute to clotting. Receptor-ligand pairs with diverse kinetic and mechanical characteristics work synergistically to arrest rapidly flowing cells on an injured vessel. Variations in hydrodynamic stresses switch on and off the function of key clotting polymers. Protein transport to, from, and within a developing clot determines whether and how fast it grows. We review ongoing experimental and modeling research to understand these and related phenomena.

To cite this article: Neeves KB, Maloney SF, Fong KP, Schmaier AA, Kahn ML, Brass LF, Diamond SL. Microfluidic focal thrombosis model for measuring murine platelet deposition and stability: PAR4 signaling enhances shear-resistance of platelet aggregates. J Thromb Haemost 2008; 6:Summary. 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 lL of whole blood and controlled flow exposure. Methods: Microfluidic methods were used for patterning acid-soluble collagen in 100 lm · 100 lm 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 a 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 Ôshear step-upÕ to 8000 s )1. Results: Wild-type murine platelets adhered and aggregated on collagen in a biphasic sheardependent 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 a 2 b 1 integrin. Those integrin a 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 thrombindependent pathways such as fibrin formation.

Propulsion at the microscale requires unique strategies such as the undulating or rotating filaments that microorganisms have evolved to swim. These features however can be difficult to artificially replicate and control, limiting the ability to actuate and direct engineered microdevices to targeted locations within practical timeframes. An alternative propulsion strategy to swimming is rolling. Here we report that low-strength magnetic fields can reversibly assemble wheel-shaped devices in situ from individual colloidal building blocks and also drive, rotate and direct them along surfaces at velocities faster than most other microscale propulsion schemes. By varying spin frequency and angle relative to the surface, we demonstrate that microwheels can be directed rapidly and precisely along user-defined paths. Such in situ assembly of readily modified colloidal devices capable of targeted movements provides a practical transport and delivery tool for microscale applications, especially those in complex or tortuous geometries.

IntroductionPlaque rupture reveals tissue factor (TF) to flowing blood, resulting in coronary thrombosis and occlusion with consequent acute myocardial infarction. Despite the prevalence of this event, the critical concentration of surface tissue factor required to cause clotting at various hemodynamic conditions remains poorly defined. In addition, the existence, source(s), and functional activity of circulating levels of tissue factor are not fully resolved in health or disease. The function of circulating TF in concert with wallderived TF may depend on prevailing flow conditions. TF in a lipid surface serves as a cofactor for factor VIIa (present at ϳ 1% of the 10-nM factor VII concentration) resulting in approximately 10 5 -fold enhancement of factor Xa formation. 1 Platelet deposition may reduce access of factor X to the TF/VIIa complex formed on the damaged wall. 2,3 Elevated TF antigen and activity are detectable in human atherosclerotic lesions and are expressed by various cell types. 4 Bonderman et al 5 determined, using ex vivo plaque disruption/scraping, that the average TF site density underneath plaques is 33 pg TF/cm 2 , corresponding to approximately 6 molecules-TF/m 2 . Drake et al 6 found that in human cardiac and skeletal muscle the TF levels were 7 and 119 ng TF/mg protein, respectively. Tissue factor pathway inhibitor (TFPI) is also elevated in atherosclerotic vessels in comparison with 10 to 20 ng TFPI/cm 2 in healthy vessels. 7 Blood-borne tissue factor antigen was first reported in a system using 5-minute ex vivo perfusion of human blood over collagencoated slides, 8 a system in which fibrin deposition was blocked by inhibited factor VIIa (FVIIa i ). Collagen-activated platelets are highly procoagulant and may present factor VIIa cofactor activity susceptible to antagonism by antibodies or FVIIa i . 9-11 A recent study of 91 individuals using the Luminex assay (Austin, TX) indicated that most healthy individuals had less than 2 pM TF in plasma, 12 a value lower than the average 4 pM TF obtained from a literature survey of plasma TF levels in healthy individuals measured by enzyme-linked immunosorbent assay (ELISA). Addition of increasing amounts of subpicomolar levels of lipidated TF to corn trypsin inhibitor (CTI)-treated whole blood indicates that active TF in healthy individuals is subpicomolar, estimated to between less than 20 fM 13 and less than 200 fM. 14 Recently, rapid splicing of TF pre-mRNA and expression of TF antigen have been reported in sonicated membranes obtained from activated platelets. 15 Under flow conditions, the transfer of tissue factor may be of importance via leukocyte delivery to platelets via CD15 16 or capture of microparticles presenting TF and PSGL-1 17-19 or derived from platelets. 10 Mathematic simulations of the hemostatic response have also taken into account the importance of tissue factor site density. Kuharsky and Fogelson 3 developed a full transport-reaction coagulation model that takes into account surface-dependent reactions, transport of factors a...

Thrombin is released as a soluble enzyme from the surface of platelets and tissue-factor-bearing cells to trigger fibrin polymerization during thrombosis under flow conditions. Although isotropic fibrin polymerization under static conditions involves protofibril extension and lateral aggregation leading to a gel, factors regulating fiber growth are poorly quantified under hemodynamic flow due to the difficulty of setting thrombin fluxes. A membrane microfluidic device allowed combined control of both thrombin wall flux (10(-13) to 10(-11) nmol/mum(2) s) and the wall shear rate (10-100 s(-1)) of a flowing fibrinogen solution. At a thrombin flux of 10(-12) nmol/mum(2) s, both fibrin deposition and fiber thickness decreased as the wall shear rate increased from 10 to 100 s(-1). Direct measurement and transport-reaction simulations at 12 different thrombin flux-wall shear rate conditions demonstrated that two dimensionless numbers, the Peclet number (Pe) and the Damkohler number (Da), defined a state diagram to predict fibrin morphology. For Da < 10, we only observed thin films at all Pe. For 10 < Da < 900, we observed either mat fibers or gels, depending on the Pe. For Da > 900 and Pe < 100, we observed three-dimensional gels. These results indicate that increases in wall shear rate quench first lateral aggregation and then protofibril extension.

Interstitial fluid flow within blood clots is a biophysical mechanism that regulates clot growth and dissolution. Assuming that a clot can be modeled as a porous medium, the physical property that dictates interstitial fluid flow is the hydraulic permeability. The objective of this study was to bound the possible values of the hydraulic permeability in clots formed in vivo and present relationships that can be used to estimate clot permeability as a function of composition. A series of clots with known densities of fibrin and platelets, the two major components of a clot, were formed under static conditions. The permeability was calculated by measuring the interstitial fluid velocity through the clots at a constant pressure gradient. Fibrin gels formed with a fiber volume fraction of 0.02-0.54 had permeabilities of 1.2 × 10(-1)-1.5 × 10(-4)μm(2). Platelet-rich clots with a platelet volume fraction of 0.01-0.61 and a fibrin volume fraction of 0.03 had permeabilities over a range of 1.1 × 10(-2)-1.5 × 10(-5)μm(2). The permeability of fibrin gels and of clots with platelet volume fraction of <0.2 were modeled as an array of disordered cylinders with uniform diameters. Clots with a platelet volume fraction of >0.2 were modeled as a Brinkman medium of coarse solids (platelets) embedded in a mesh of fine fibers (fibrin). Our data suggest that the permeability of clots formed in vivo can vary by up to five orders of magnitude, with pore sizes that range from 4 to 350 nm. These findings have important implications for the transport of coagulation zymogens/enzymes in the interstitial spaces during clot formation, as well as the design of fibrinolytic drug delivery strategies.

Mitogen-activated protein kinases (MAPKs) are expressed in platelets and are activated downstream of physiological agonists. Pharmacological and genetic evidence indicate that MAPKs play a significant role in hemostasis and thrombosis, but it is not well understood how MAPKs are activated upon platelet stimulation. Here, we show that apoptosis signal-regulating kinase 1 (ASK1), a member of the MAP3K family, is expressed in both human and murine platelets. ASK1 is rapidly and robustly activated upon platelet stimulation by physiological agonists. Disruption of ( ) resulted in a marked functional defect in platelets. platelets showed an impaired agonist-induced integrin αβ activation and platelet aggregation. Although there was no difference in Ca rise, platelet granule secretion and thromboxane A (TxA) generation were significantly attenuated in platelets. The defective granule secretion observed in platelets was a consequence of impaired TxA generation. Biochemical studies showed that platelet agonists failed to activate p38 MAPK in platelets. On the contrary, activation of c-Jun-terminal kinases and extracellular signal-regulated kinase 1/2 MAPKs was augmented in platelets. The defect in p38 MAPK results in failed phosphorylation of cPLA in platelets and impaired platelet aggregate formation under flow. The absence of Ask1 renders mice defective in hemostasis as assessed by prolonged tail-bleeding times. Deletion of also reduces thrombosis as assessed by delayed vessel occlusion of carotid artery after FeCl-induced injury and protects against collagen/epinephrine-induced pulmonary thromboembolism. These results suggest that the platelet Ask1 plays an important role in regulation of hemostasis and thrombosis.