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•Less-deformable sickle red cells marginate toward blood vessel walls, leading to altered local wall shear stress and endothelial dysfunction.•Pathological biophysical alterations in sickle RBCs cause endothelial dysfunction independently from vaso-occlusion and adhesion.

Prior reports indicate that the convex membrane curvature of phosphatidylserine (PS)-containing vesicles enhances formation of binding sites for factor Va and lactadherin. Yet, the relationship of convex curvature to localization of these proteins on cells remains unknown. We developed a membrane topology model, using phospholipid bilayers supported by nano-etched silica substrates, to further explore the relationship between curvature and localization of coagulation proteins. Ridge convexity corresponded to maximal curvature of physiologic membranes (radii of 10 or 30 nm) and the troughs had a variable concave curvature. The benchmark PS probe lactadherin exhibited strong differential binding to the ridges, on membranes with 4% to 15% PS. Factor Va, with a PS-binding motif homologous to lactadherin, also bound selectively to the ridges. Bound factor Va supported coincident binding of factor Xa, localizing prothrombinase complexes to the ridges. Endothelial cells responded to prothrombotic stressors and stimuli (staurosporine, tumor necrosis factor-α [TNF- α]) by retracting cell margins and forming filaments and filopodia. These had a high positive curvature similar to supported membrane ridges and selectively bound lactadherin. Likewise, the retraction filaments and filopodia bound factor Va and supported assembly of prothrombinase, whereas the cell body did not. The perfusion of plasma over TNF-α–stimulated endothelia in culture dishes and engineered 3-dimensional microvessels led to fibrin deposition at cell margins, inhibited by lactadherin, without clotting of bulk plasma. Our results indicate that stressed or stimulated endothelial cells support prothrombinase activity localized to convex topological features at cell margins. These findings may relate to perivascular fibrin deposition in sepsis and inflammation.

•Human, canine, ovine, and porcine platelets exhibit disparate biophysical signatures, whereas human and murine platelets are similar.•Multiple biophysical parameters integrate during clot formation, measured by bulk clot contraction, and attenuate biophysical differences.

Abnormal sickle red blood cell (sRBC) biomechanics, including pathological deformability and adhesion, correlate with clinical severity in sickle cell disease (SCD). Clinical intravenous fluids (IVFs) of various tonicities are often used during treatment of vaso-occlusive pain episodes (VOE), the major cause of morbidity in SCD. However, evidence-based guidelines are lacking, and there is no consensus regarding which IVFs to use during VOE. Further, it is unknown how altering extracellular fluid tonicity with IVFs affects sRBC biomechanics in the microcirculation, where vaso-occlusion takes place. Here, we report how altering extracellular fluid tonicity with admixtures of clinical IVFs affects sRBC biomechanical properties by leveraging novel in vitro microfluidic models of the microcirculation, including 1 capable of deoxygenating the sRBC environment to monitor changes in microchannel occlusion risk and an “endothelialized” microvascular model that measures alterations in sRBC/endothelium adhesion under postcapillary venular conditions. Admixtures with higher tonicities (sodium = 141 mEq/L) affected sRBC biomechanics by decreasing sRBC deformability, increasing sRBC occlusion under normoxic and hypoxic conditions, and increasing sRBC adhesion in our microfluidic human microvasculature models. Admixtures with excessive hypotonicity (sodium = 103 mEq/L), in contrast, decreased sRBC adhesion, but overswelling prolonged sRBC transit times in capillary-sized microchannels. Admixtures with intermediate tonicities (sodium = 111-122 mEq/L) resulted in optimal changes in sRBC biomechanics, thereby reducing the risk for vaso-occlusion in our models. These results have significant translational implications for patients with SCD and warrant a large-scale prospective clinical study addressing optimal IVF management during VOE in SCD.

Ex vivo gene therapy using lentiviral vectors (LVs) is a proven approach to treat and potentially cure many hematologic disorders and malignancies but remains stymied by cumbersome, cost-prohibitive, and scale-limited production processes that cannot meet the demands of current clinical protocols for widespread clinical utilization. However, limitations in LV manufacture coupled with inefficient transduction protocols requiring significant excess amounts of vector currently limit widespread implementation. Herein, we describe a microfluidic, mass transport-based approach that overcomes the diffusion limitations of current transduction platforms to enhance LV gene transfer kinetics and efficiency. This novel ex vivo LV transduction platform is flexible in design, easy to use, scalable, and compatible with standard cell transduction reagents and LV preparations. Using hematopoietic cell lines, primary human T cells, primary hematopoietic stem and progenitor cells (HSPCs) of both murine (Sca-1+) and human (CD34+) origin, microfluidic transduction using clinically processed LVs occurs up to 5-fold faster and requires as little as one-twentieth of LV. As an in vivo validation of the microfluidic-based transduction technology, HSPC gene therapy was performed in hemophilia A mice using limiting amounts of LV. Compared to the standard static well-based transduction protocols, only animals transplanted with microfluidic-transduced cells displayed clotting levels restored to normal.

We report a cell-mediated, targeted drug delivery system utilizing polyelectrolyte multilayer capsules that hybridize with the patient’s own platelets upon intravenous administration. The hybridized platelets function as the sensor and actuator for targeted drug delivery and controlled release in our system. These capsules are biochemically and mechanically tuned to enable platelet adhesion and capsule rupture upon platelet activation and contraction, enabling the targeted and controlled “burst” release of an encapsulated biotherapeutic. As platelets are the “first responders” in the blood clot formation process, this platelet-hybridized system is ideal for the targeted delivery of clot-augmenting biotherapeutics wherein immediate therapeutic efficacy is required. As proof-of-concept, we tailored this system to deliver the pro-clotting biotherapeutic factor VIII for hemophilia A patients that have developed inhibitory antifactor VIII antibodies. The polyelectrolyte multilayer capsules physically shield the encapsulated factor VIII from the patient’s inhibitors during circulation, preserving its bioactivity until it is delivered at the target site viaplatelet contractile force. Using an in vitromicrofluidic vascular injury model with factor VIII-inhibited blood, we demonstrate a 3.8× increase in induced fibrin formation using capsules loaded with factor VIII at a concentration an order of magnitude lower than that used in systemic delivery. We further demonstrate that clot formation occurs 18 min faster when factor VIII loaded capsules are used compared to systemic delivery at the same concentration. Because platelets are integral in the pathophysiology of thrombotic disorders, cancer, and innate immunity, this paradigm-shifting smart drug delivery system can be similarly applied to these diseases.

Haemostasis occurs at sites of vascular injury, where flowing blood forms a clot, a dynamic and heterogeneous fibrin-based biomaterial. Paramount in the clot’s capability to stem haemorrhage are its changing mechanical properties, the major drivers of which are the contractile forces exerted by platelets against the fibrin scaffold. However, how platelets transduce microenvironmental cues to mediate contraction and alter clot mechanics is unknown. This is clinically relevant, as overly softened and stiffened clots are associated with bleeding and thrombotic disorders. Here, we report a high-throughput hydrogel-based platelet-contraction cytometer that quantifies single-platelet contraction forces in different clot microenvironments. We also show that platelets, via the Rho/ROCK pathway, synergistically couple mechanical and biochemical inputs to mediate contraction. Moreover, highly contractile platelet subpopulations present in healthy controls are conspicuously absent in a subset of patients with undiagnosed bleeding disorders, and therefore may function as a clinical diagnostic biophysical biomarker.

The mechanism of action of the widely used in vivo ferric chloride (FeCl3) thrombosis model remains poorly understood; although endothelial cell denudation is historically cited, a recent study refutes this and implicates a role for erythrocytes. Given the complexity of the in vivo environment, an in vitro reductionist approach is required to systematically isolate and analyze the biochemical, mass transfer, and biological phenomena that govern the system. To this end, we designed an “endothelial-ized” microfluidic device to introduce controlled FeCl3 concentrations to the molecular and cellular components of blood and vasculature. FeCl3 induces aggregation of all plasma proteins and blood cells, independent of endothelial cells, by colloidal chemistry principles: initial aggregation is due to binding of negatively charged blood components to positively charged iron, independent of biological receptor/ligand interactions. Full occlusion of the microchannel proceeds by conventional pathways, and can be attenuated by antithrombotic agents and loss-of-function proteins (as in IL4-R/Iba mice). As elevated FeCl3 concentrations overcome protective effects, the overlap between charge-based aggregation and clotting is a function of mass transfer. Our physiologically relevant in vitro system allows us to discern the multifaceted mechanism of FeCl3-induced thrombosis, thereby reconciling literature findings and cautioning researchers in using the FeCl3 model.

Although the biology of platelet adhesion on subendothelial matrix after vascular injury is well characterized, how the matrix biophysical properties affect platelet physiology is unknown. Here we demonstrate that geometric orientation of the matrix itself regulates platelet α-granule secretion, a key component of platelet activation. Using protein microcontact printing, we show that platelets spread beyond the geometric constraints of fibrinogen or collagen micropatterns with <5-µm features. Interestingly, α-granule exocytosis and deposition of the α-granule contents such as fibrinogen and fibronectin were primarily observed in those areas of platelet extension beyond the matrix protein micropatterns. This enables platelets to “self-deposit” additional matrix, provide more cellular membrane to extend spreading, and reinforce platelet-platelet connections. Mechanistically, this phenomenon is mediated by actin polymerization, Rac1 activation, and αIIbβ3 integrin redistribution and activation, and is attenuated in gray platelet syndrome platelets, which lack α-granules, and Wiskott-Aldrich syndrome platelets, which have cytoskeletal defects. Overall, these studies demonstrate how platelets transduce geometric cues of the underlying matrix geometry into intracellular signals to extend spreading, which endows platelets spatial flexibility when spreading onto small sites of exposed subendothelium.

Monocyte transmigration across stimulated ECs promotes further monocyte recruitment, inhibits monocyte apoptosis, and leads to a reduction in antimicrobial protein expression. We investigated the hypothesis that transmigration drives monocyte transcriptional changes. Using Agilent whole human genome microarrays, we identified over 692 differentially expressed genes (2×, P<0.05) in freshly isolated human monocytes following 1.5 h of transmigration across IL‐1β‐stimulated ECs compared with untreated monocytes. Genes up‐regulated by monocyte transmigration belong to a number of over‐represented functional groups including immune response and inhibition of apoptosis. qRT‐PCR confirmed increased expression of MCP‐1 and −3 and of NAIP following monocyte transmigration. Additionally, quantification of Annexin V binding revealed a reduction in apoptosis following monocyte transmigration. Comparison of gene expression in transmigrated monocytes with additional controls (monocytes that failed to transmigrate and monocytes incubated beneath stimulated ECs) revealed 89 differentially expressed genes, which were controlled by the process of diapedesis. Functional annotation of these genes showed down‐regulation of antimicrobial genes (e.g., α‐defensin down 50×, cathelicidin down 9×, and CTSG down 3×). qRT‐PCR confirmed down‐regulation of these genes. Immunoblots confirmed that monocyte diapedesis down‐regulates α‐defensin protein expression. However, transmigrated monocytes were functional and retained intact cytokine and chemokine release upon TLR ligand exposure. Overall, these data indicate that the process of monocyte transmigration across stimulated ECs promotes further monocyte recruitment and inhibits monocyte apoptosis. Unexpectedly, following transmigration, monocytes displayed reduced antimicrobial protein expression.

Hyperleukocytosis, most commonly defined as a white blood cell (WBC) count > 100,000/μL, is an oncologic emergency in acute leukemia that can lead to leukostasis, which occurs when leukemia cells obstruct the microvasculature resulting in significant morbidity and mortality from neurologic (CNS hemorrhage, thrombosis) or pulmonary (respiratory distress, hypoxia) symptoms. The underlying mechanisms are poorly understood but are thought to be related to increased blood viscosity, secondary to high WBC count, leukemia cell aggregation, and the abnormal mechanical properties, size, and cell-cell interactions of leukemia cells. Leukapheresis is a commonly used therapy for rapid cytoreduction in symptomatic patients, but the procedure is not without risks. No existing methods reliably predict leukostasis or guide treatment including the commonly used WBC count, which only loosely correlates with leukostasis and does not accurately describe the blood viscosity at the microvascular level. Importantly, while hematocrit/hemoglobin levels (Hgb) are known to be major contributors to blood viscosity, they have not been systematically assessed in leukostasis risk, and Hgb often decreases as leukemic cell counts rise, complicating the issue. Incorporating Hgb levels may better predict leukostasis and assist physicians balancing the risk of hyperleukocytosis compared to the interventions themselves.

Pulsations in blood flow alter gene and protein expressions in endothelial cells (EC). A computer-controlled system was developed to mimic the common carotid artery flow waveform and shear stress levels or to provide steady flow of the same mean shear stress in a parallel plate flow chamber. The pseudo-steady state shear stress was determined from real-time pressure gradient measurements and compared to the Navier–Stokes equation solution. Following 24 h of steady flow (SF: 13 dyne/cm2), pulsatile arterial flow (AF: average=13 dyne/cm2, range=7–25 dyne/cm2) or static conditions, heme oxygenase-1 (HO-1) and prostaglandin H synthase-2 (PGHS-2) mRNA and protein expressions from human umbilical vein endothelial cells were measured. Relative to steady flow, pulsatile arterial flow significantly attenuated mRNA upregulation of HO-1 (SF: 7.26±2.70-fold over static, AF: 4.84±0.37-fold over static; p< 0.01) and PGHS-2 (SF: 6.11±1.79-fold over static, AF: 3.54±0.79-fold over static; p< 0.001). Pulsatile arterial flow (4.57±0.81-fold over static, p< 0.01) also significantly reduced the steady-flow-induced HO-1 protein upregulation (7.99±1.29-fold over static). These findings reveal that EC can discriminate between different flow patterns of the same average magnitude and respond at the molecular level.Pulsations in blood flow alter gene and protein expressions in endothelial cells (EC). A computer-controlled system was developed to mimic the common carotid artery flow waveform and shear stress levels or to provide steady flow of the same mean shear stress in a parallel plate flow chamber. The pseudo-steady state shear stress was determined from real-time pressure gradient measurements and compared to the Navier–Stokes equation solution. Following 24 h of steady flow (SF: 13 dyne/cm2), pulsatile arterial flow (AF: average=13 dyne/cm2, range=7–25 dyne/cm2) or static conditions, heme oxygenase-1 (HO-1) and prostaglandin H synthase-2 (PGHS-2) mRNA and protein expressions from human umbilical vein endothelial cells were measured. Relative to steady flow, pulsatile arterial flow significantly attenuated mRNA upregulation of HO-1 (SF: 7.26±2.70-fold over static, AF: 4.84±0.37-fold over static; p< 0.01) and PGHS-2 (SF: 6.11±1.79-fold over static, AF: 3.54±0.79-fold over static; p< 0.001). Pulsatile arterial flow (4.57±0.81-fold over static, p< 0.01) also significantly reduced the steady-flow-induced HO-1 protein upregulation (7.99±1.29-fold over static). These findings reveal that EC can discriminate between different flow patterns of the same average magnitude and respond at the molecular level.

Phenotypic modulation of vascular smooth muscle cells (VSMC) and reactive oxygen species (ROS) is important in vascular pathogenesis. Understanding how these factors relate to cell migration can improve design of therapeutic interventions to control vascular disease. We compared the proliferation, protein content and migration of cultured aortic VSMC from wild type (WT) versus transgenic mice (Tgp22phox), in which overexpression of p22phox was targeted to VSMC. Also, we compared H2O2 generation and expression of specific phenotypic markers of non-migrating with migrating WT versus Tgp22phox VSMC in an in vitro wound scratch model. Enhanced H2O2 production in Tgp22phoxversus WT VSMC (p < 0.005) significantly correlated with increased protein content, proliferation, and migration. VSMC migrating across the wound edge produced more H2O2 than non-migrating VSMC (p < 0.05). The expression of synthetic phenotypic markers, tropomyosin 4 and myosin heavy chain embryonic (SMemb), was enhanced significantly, while the expression of contractile marker, smooth muscle α-actin, was reduced significantly in migrating versus non-migrating cells, and also in Tgp22phoxversus WT (p < 0.005) VSMC. These results are consistent with increased production of ROS accelerating the switch from the contractile to the synthetic phenotype, characterized by increases in proliferation, migration, and expression of TM4 and SMemb and decreased α-actin.

Inflammation-mediated endothelial cell (EC) dysfunction likely contributes to the pathogenesis of several vascular diseases including atherosclerosis. We found that stimulation of human umbilical vein ECs with lipopolysaccharide induced secretion of cyclophilin (CyPA) an intracellular protein belonging to the immunophilin family. We then found that when added exogenously CyPA has direct effects on ECs in vitro. At low concentrations (10 to 100 ng/ml) CyPA increased EC proliferation, migration, invasive capacity, and tubulogenesis. Gelatin zymography indicated increased secretion of active matrix metalloproteinase-2, a mediator of cell migration and angiogenesis. At high concentrations (eg, 2 μg/ml) CyPA had opposite effects, decreasing EC migration and viability, possibly in relation to induction of Toll-like receptor-4 expression, detected by immunocytochemistry and flow cytometry. In vivoCyPA expression was not detectable in the luminal ECs of normal mouse carotid arteries but was rapidly induced after systemic lipopolysaccharide injection. In an experimental mouse model of atherosclerosis, CyPA expression was detected in the ECs of neocapillaries of carotid artery lesions, supporting its association with pathological angiogenesis suggested by our in vitroresults. In conclusion, we found that CyPA has a biphasic activity on ECs in vitroand is up-regulated in vivoin ECs under pathological states. Our results suggest that CyPA is a novel paracrine and autocrine modulator of EC functions in immune-mediated vascular disease.

Prior reports indicate that the convex membrane curvature of phosphatidylserine (PS)-containing vesicles enhances formation of binding sites for factor Va and lactadherin. Yet, the relationship of convex curvature to localization of these proteins on cells remains unknown. We developed a membrane topology model, using phospholipid bilayers supported by nano-etched silica substrates, to further explore the relationship between curvature and localization of coagulation proteins. Ridge convexity corresponded to maximal curvature of physiologic membranes (radii of 10 or 30 nm) and the troughs had a variable concave curvature. The benchmark PS probe lactadherin exhibited strong differential binding to the ridges, on membranes with 4% to 15% PS. Factor Va, with a PS-binding motif homologous to lactadherin, also bound selectively to the ridges. Bound factor Va supported coincident binding of factor Xa, localizing prothrombinase complexes to the ridges. Endothelial cells responded to prothrombotic stressors and stimuli (staurosporine, tumor necrosis factor-α [TNF- α]) by retracting cell margins and forming filaments and filopodia. These had a high positive curvature similar to supported membrane ridges and selectively bound lactadherin. Likewise, the retraction filaments and filopodia bound factor Va and supported assembly of prothrombinase, whereas the cell body did not. The perfusion of plasma over TNF-α–stimulated endothelia in culture dishes and engineered 3-dimensional microvessels led to fibrin deposition at cell margins, inhibited by lactadherin, without clotting of bulk plasma. Our results indicate that stressed or stimulated endothelial cells support prothrombinase activity localized to convex topological features at cell margins. These findings may relate to perivascular fibrin deposition in sepsis and inflammation.

Hyperleukocytosis, most commonly defined as a white blood cell (WBC) count greater than 100,000/μL, is an emergency in acute leukemia, possibly resulting in life-threatening microvascular obstruction, or leukostasis, leading to neurologic (CNS hemorrhage, thrombosis) or pulmonary (respiratory distress, hypoxia) complications. The underlying mechanisms remain poorly understood and are canonically attributed to blood hyperviscosity secondary to high WBC count and abnormal biophysical properties of leukemia cells themselves (leukemia immunophenotype, increased cell size, adhesion, and stiffness). Leukapheresis is a commonly-used therapy for rapid cytoreduction in symptomatic patients, but the procedure carries risk and existing guidelines are supported by scant evidence. Interestingly, despite hematocrit(Hct)/hemoglobin(Hgb) levels being major drivers of blood viscosity due to the high prevalence of circulating red cells (RBCs), how Hct/Hgb mediates hyperviscosity in acute leukemia is unknown. This is clinically important as Hct/Hgb often decrease as leukemic cell counts rise, and acute leukemia patients with anemia are often transfused. While sickle cell disease guidelines advise using a target post transfusion Hct of 30% to minimize iatrogenic hyperviscosity and its morbid complications, no guidelines have been established for acute leukemia. As such, can RBC transfusion actually increase leukostasis risk in acute leukemia?

A subset of patients with chronic bleeding remain undiagnosed even after extensive diagnostic evaluation are labeled as “bleeding of unknown cause” (BUC). The key barrier to treating these patients is that they have a clinical bleeding tendency in the presence of normal diagnostic tests, and optimal methods for monitoring and treating patients with BUC remain unknown. While patients with BUC have symptoms of a primary hemostatic disorder, there is no diagnostic test or biomarker that can accurately identify which patients are at risk for bleeding such as those with mild Von Willebrand Disease (VWD) which comprise a broad spectrum of patients with varying degrees of bleeding. In order to fill this diagnostic gap in disorders of primary hemostasis, there is a clinical need for more assays of platelet function.

The article offers step-by-step instructions for knitting the Hillary Vest designed by Yumiko Sakurai.

Carden: GBT: Honoraria; NIH: Research Funding. Mannino:Sanguina, LLC: Employment, Equity Ownership. Lam:Sanguina, LLC: Equity Ownership.

Background:Treatment of thrombosis relies on prompt resolution of thrombi to restore blood flow to avoid ischemic injury. However, our understanding of the step-by-step process of how thromboses resolve remains limited due in large part to the lack of sufficient technologies. In addition, as thromboses require days to weeks to resolve, existing in vitroand in vivosystems cannot monitor this process, especially in the microvasculature where thrombi are difficult to visualize. As such, questions such as how do the cellular and biochemical composition of a clot change as it resolves and how do hemodynamics affect this process remain unanswered. This is particularly important in thromboinflammatory conditions such as autoimmune/inflammatory disorders in which patients are chronically at risk for microvascular thromboses but the pathophysiology and therefore optimal therapies remain unclear. Thus, a pressing need exists for an assay that assesses how microvascular thromboses resolve over long time scales, especially under thromboinflammatory conditions. We recently developed a microfluidic system that assesses microvascular events, including endothelial dysfunction and permeability, in response to proinflammatory signals over months (Qiu, Nature Biomed Eng. 2018). Here we leverage this system to monitor not only how microvascular thromboses form but, importantly, how they resolve over long timescales. Moreover, our system also enables the monitoring of how antithrombotic drugs and anticoagulants “work” in the context of existing inflammatory thrombi, which will provide insight into the pathophysiology as well as provide evidence for the use of different therapies.

Jobe: CSL: Consultancy; Shire: Consultancy; Octapharma: Consultancy.

No relevant conflicts of interest to declare.

Meeks: Biogen: Membership on an entity's Board of Directors or advisory committees; Genentech: Membership on an entity's Board of Directors or advisory committees; Bayer Healthcare: Membership on an entity's Board of Directors or advisory committees; Grifols: Membership on an entity's Board of Directors or advisory committees; CSL Behring: Membership on an entity's Board of Directors or advisory committees; Shire: Membership on an entity's Board of Directors or advisory committees. Lam:Sanguina, LLC: Equity Ownership.

No relevant conflicts of interest to declare.

Jobe: Bayer: Membership on an entity's Board of Directors or advisory committees; Biogen: Membership on an entity's Board of Directors or advisory committees; CSL-Behring: Membership on an entity's Board of Directors or advisory committees.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

Background:Recent clinical trials have demonstrated the efficacy and safety of gene therapy utilizing HIV-derived lentiviral vectors (LVs) for blood disorders. However, the LV requirements and clinical ex vivocell transduction protocols used in these studies exposes the limitations of the technology and beckons the need for improved LV manufacturing and clinical transduction efficiency. Many methods have been devised to enhance efficiency, although none have circumvented the exorbitant amounts of virus required to achieve therapeutic HSC transduction. Furthermore, prolonged ex vivocell culture is necessary to achieve sufficient transduction despite exposure to toxic byproducts of LV production. To that end, we developed a novel, scalable microfluidic for clinical LV transduction that leverages mass transfer principles to significantly reduce the amount of LV required to achieve therapeutic levels of gene transfer and transduction time by more efficiently exposing cells to virus.

No relevant conflicts of interest to declare.

Introduction:While the platelet’s role in achieving hemostasis in the context of bleeding is well-characterized, how platelets support and maintain vascular integrity during homeostatic, non-thrombotic conditions remains unclear. Previous studies have shown that single platelets fill micron/submicron-sized “gaps” of small discontinuities in the endothelium. In this context, however, as the overall force of adhesion correlates with the area of exposed subendothelial matrix, how platelets establish sufficient adhesion to resist the dynamic shear forces of the circulation are unknown. In this study, we investigate, using protein microcontact printing, how platelets sense the geometry of the matrix micro/nano-environment and transduce those cues to affect platelet function. Our results reveal that platelets, upon sensing spatially-limited (<25 µm2area) micro/nanopatterns of fibrinogen or collagen, spatially regulate actin-rich filopodia extension, via Rac1, beyond the geometric constraints of the matrix pattern, redistribute α-granules to enable “self-deposition” of fibrinogen/fibronectin matrix on those regions, and spread onto those newly deposited matrix via integrin αIIbβ3-mediated interactions. Furthermore, this phenomenon is markedly attenuated in Gray Platelet Syndrome platelets, which lack α-granules, and Wiskott-Aldrich Syndrome platelets, which have impaired integrin αIIbβ3-mediated activation, decreased α-granules, and cytoskeletal defects.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

Whereas surface-immobilized fibrinogen readily causes platelet adhesion and spreading, soluble fibrinogen, on the other hand, does not lead to platelet activation without the presence of other hemostatic/thrombotic signals. This dramatically different response of platelets to fibrinogen may be due to biochemical difference in fibrinogen unfolding (Agnihotri, et al., Langmuir, 2004), but may also be due to the difference in how platelets directly react to the physical properties of the substrate underneath the fibrinogen. Similarly, recent studies have shown that many types of adherent eukaryotic cells respond differently to substrates of different physical properties. In particular, cells are able to mechanosense the stiffness of their underlying substrate, and to change their spreading, re-organize their cytoskeleton, and even alter gene expression responding to the sensed stiffness (Discher, et al., Science, 2005). More specific to platelets, our group has recently shown that the contraction of single platelets on fibrinogen surfaces increases with increasing substrate stiffness (Lam, et al., Nat Mater, 2011). Therefore, we hypothesize that substrate stiffness can also affect platelet adhesion and spreading on fibrinogen. In this study, we synthesized polyacrylamide (PAA) gels, which can be tuned to different stiffnesses and easily modified with covalently-bound fibrinogen on the surface. This assay enables independent control of substrate stiffness while maintaining constant biochemical composition and fibrinogen density, and we applied this system to quantitatively investigate the role of substrate stiffness in platelet adhesion and spreading on fibrinogen.3 μg/ml fibrinogen was covalently bound to the surface of PAA gels of different stiffnesses (0.25, 0.5, 2.5, 5, 50 and 100 kilopascals (KPa)) (Figure 1A). This applied range of substrate stiffness mimics the stiffness of different tissues in the body (Engler, et al., Cell, 2006). Moreover, glass (with a stiffness of ∼65–70 × 103 KPa) adsorbed with 3 μg/ml fibrinogen was also used for comparison. During 2 hour incubations, washed human platelets differently adhered to and spread on the surface of PAA gels. By simply varying the stiffness of the fibrinogen-bound PAA gels, we observed dramatic differences in the number of adherent platelets and their morphology (Figure 1B). The number of adherent platelets increased with increasing stiffness, reaching a plateau at 2.5 KPa, with adherence similar to that of fibrinogen-adsorbed glass (Figure 1C). While platelets did not spread on 2.5 KPa and softer gels, approximately 30–40% adherent platelets spread on 5KPa and stiffer gels, resulting in a significantly higher average spreading area of adherent platelets (Figure 1D and E). However, compared to all the gels, mostly all platelets adhered on glass surface spread (area > 35 μm2) and showed a significantly higher spreading area (Figure 1D and E). Moreover, no significant difference in fibrinogen concentration was detected among fibrinogen-bound PAA gels of different stiffnesses and fibrinogen-adsorbed glass (Data not shown), which indicated that the difference we observed could be independently due to the substrate stiffness.Our data suggest that platelets sense the mechanical properties of the underlying substrate to fine-tune the degree of adhesion and spreading on fibrinogen. Thus, fibrinogen on soft substrates appears to activate platelets to a lesser degree than the same concentration of fibrinogen on stiffer substrates. We are currently investigating how substrate stiffness triggers mechanotransduction in platelets and affects their outside-in activation and signaling. Our study also provides potential insights for preventing clot formation on implanted biomaterials and medical devices.No relevant conflicts of interest to declare.

Abstract 3300

Hemostatic and thrombotic processes are dependent on platelets, coagulation factors, endothelial cells and hemodynamic flow. Current in vitro assays, however, only encompass one or two of these variables, rendering their results difficult to extrapolate to the in vivo setting. To that end, we have further refined our previously published endothelialized microfluidic system for studying thrombotic processes (Tsai et al, JCI, 2012) to specifically incorporate simultaneous differential flow rates spanning venous (10 s−1), capillary (100 s−1), and arterial/arteriolar (1000 s−1) flow conditions. Overall, key advantages of our system include: 1) successful integration of whole blood, an intact endothelium, and hemodynamic flow in a single microfluidic device, 2) simultaneous differential flow rates in a single experiment spanning 3 orders of magnitude, 3) use of corn trypsin inhibitor (CTI) as the sole anticoagulant, enabling calcium dependent processes to occur, and 4) minimal sample volume (1–2 mL) even for high shear conditions. We have applied our microsystem to elucidate some of the underlying mechanistic differences between venous and arterial thrombosis.We used photolithographic and microfabrication techniques. We previously employed to develop the silicone-based microfluidic device and applied our optimized protocol to culture human endothelial cells (HUVECs) to confluency throughout the entire inner surfaces of the system (Tsai et al, JCI, 2012 and Myers et al, JoVE, 2012) (Figure 1A, 1B). Once HUVECs are successfully cultured, whole blood with 5 % v/v fluorescently-labeled fibrinogen and cell membrane dyes is flowed into the microfluidic system. Our device can then be “activated” to induce 3 different simultaneous shear conditions (10 s−1, 100 s−1 and 1000 s−1) in 3 separate endothelialized microchannels by differentially varying the hydrodynamic resistance in each microchannel. Figure 1C shows a fibrin network that is formed under flow conditions (with whole blood anticoagulated with CTI) in one of the endothelialized microchannels.Elevated levels of prothrombin are known to increase risk of venous, but not arterial thrombosis. We applied our novel system to investigate whether differences between arterial and venous thrombosis risk depend, at least in part, on differences in blood flow/shear in those vessels. By adding 1.38 μM of prothrombin to whole blood with CTI and flowing it into our system, we observed platelet-rich and fibrin rich thrombi form and occlude the 10 s−1 and 100 s−1 microchannels, but not the 1000 s−1 microchannel. In contrast, whole blood with CTI without prothrombin yielded minimal fibrin formation on the endothelial cells with no platelet adhesion/aggregation detected in all 3 shear conditions.Our results suggest that arterial and venous thrombosis risk is due, at least in part, to the differences in shear flow and highlights the utility of our novel endothelialized microfluidic system with simultaneous differential flow rates. Coupled with complementary in vivo experiments, our system is a powerful tool to investigate the underlying mechanisms of hemostatic and thrombotic processes involving flow.No relevant conflicts of interest to declare.

Electricity has historically been used in medical applications such as defibrillators, cauterization, and electrosurgery. Additionally, Kalghatgi et al. [1] demonstrate that high voltage electrical fields (∼30 kilo volts) activate platelets and induce coagulation. However, the exact effect of applied electrical current on clot formation is unknown. We show that upon direct application of electricity (voltages between 5 – 40 volts), platelets are activated immediately and clots rapidly form without excessive heating. This newfound application to induce blood clot formation may enable a new and novel class of therapeutics to achieve hemostasis at sites of bleeding. As newer hemostatic agents are currently derived from animal or human products, which carry risk of blood borne infections and immune dysregulation, a clear need exists for novel therapies to achieve hemostasis.Our experimental setup consisted of a silicone polydimethylsiloxane (PDMS) chamber with embedded metallic wires (Figure 1A). The metallic leads were connected to an Agilent E3649A variable power supply and a constant voltage was applied to the chamber for 1 minute. A Fluke 179 multimeter monitored the temperature with a thermocouple lead inserted into the liquid in the chamber. Experiments were conducted using whole human blood, platelet rich plasma (PRP), platelet poor plasma (PPP), and isolated, washed platelets. Additionally, fluorescently labeled fibrinogen (Alexa Fluor 488) was added to the blood product, 5%v/v, to assess fibrin formation.Initial experiments characterized the electrical characteristics of the different components of blood pertinent for clot formation to assess the potential safety concerns. Voltages between 5 – 40 volts (V) result in currents below 0.1 amps (A) and temperatures between 20 – 50°C. Nominally, current values of 0.1A and greater are considered deadly [2], and thermal tissue damage caused by temperatures below 45°C are considered reversible [2], therefore the majority of our work focuses on voltages less than 30V. Figure 1B shows the timeline of fibrin network formation for a control whole blood sample versus a whole blood sample exposed to 30V for 60 seconds. At 30V and immediately after electrical stimulus, platelet aggregation begins to form. At 120 seconds, fibrin polymerization initiates and showed complete coverage at 180 seconds, as well as numerous clusters of platelet aggregates. In the absence of electrical stimulus, no fibrin polymerization or platelet aggregation was detected until greater than 240 seconds and full network coverage was complete by 420 second. Platelet aggregration was more pronouced with electrical stimulus, as compared to the control case. Various voltages were tested with the mean time to complete the fibrin network formation in stimulated and unstimulated whole blood was 170 seconds vs. 320 seconds, respectively (n = 3) indicating a 53% increase in fibrin formation and platelet aggregation. Additional experiments were conducted on anti-coagulated PPP, PRP, and isolated, washed platelets showing no evidence of fibrin polymerization. This suggests that all the components in blood are necessary to create the fibrin scaffold when exposed to electricity. Continued work will focus on unraveling the underlying mechanisms of how electrical stimulation affects platelet aggregation and coagulation.Our results suggest that direct electrical stimulation promotes clot formation and could potentially lead to a new category of hemostatic therapies that are free from the infectious risks and immune effects that encumber current human or animal-derived agents. With the addition of electrical stimulus, fibrin networks form on average 53% faster than control conditions. We anticipate this concept of applying electricity to different processes in the blood will have significant implications for experimental and clinical hematology.No relevant conflicts of interest to declare.

Previous studies on clot formation have shown that the mechanical properties of clots have direct effects on hemostasis and thrombosis, and alterations of those clot mechanics are associated with disease(Collet, et al. 2006) (Hvas, et al. 2007). As such, understanding the mechanical properties of clots is vital to understand hemostasis and thrombosis. As platelets drive this contraction phenomenon, single platelet measurements are required to obtain a mechanistic understanding of the retraction process and to identify specific therapeutic targets for disease states in which platelet/clot retraction is pathologically altered. In addition, as fibrin has recently been shown to have extremely complex material and mechanical properties (Brown, et al. 2009), single platelet studies would decouple the effects of fibrin from platelets when examining clot mechanics. However, few studies have focused on the biomechanical role of platelets in clot formation and clot mechanics, especially at the single cell level. Our group has recently published measurements of single platelet contraction (Lam, et al, Nature Mat, 2011), showing that platelets are capable of applying large forces and are quite varied in their response. However, the key barrier which has prevented the study of single platelets has been the lack of a technology with the sufficient precision and sensitivity to both manipulate and measure individual platelets in a high throughput manner. To that end, we have extended a technique (Polio, et al. 2012) that is capable of measuring the contraction of individual platelets in a high throughput manner.Here we precisely pattern FITC conjugated fibrinogen dots in a geometrical array (Fig 1A) on polyacrylamide (PAA) gels. Thrombin activated platelets are incubated on the gel and contract upon contact with the micropatterned fibrinogen “dots”. When the platelet comes into contact with two dots and contracts, the distance in which the platelet moves the dots from their original position is used to determine the force. Conceptually, this is similar to the idea of a linear spring, in which a certain spring displacement corresponds to a known force. Using this technique, we measured 71 platelets which were attached to two fibrinogen dots each, and found that on low stiffness gels, that the average contractile force was approximately 4nN (Fig 2A). Platelets may attach to a maximum of four dots, but do so with a much lower frequency as compared to two dots (Fig 2B). Preliminary results indicate that as platelet area increases, as indicated by contact with additional protein “dots”, the total force exerted by the platelet increases, with a maximum contractile force achieved when touching three protein dots (Fig 2C). Based on this data, there may be an optimum platelet spread area that maximizes contractile force.We will determine how the biophysical parameters, such as micro-environmental stiffness and shear flow, quantitatively affect platelet contractility. As our current understanding of the underlying biological mechanisms of platelet contraction is solely qualitative, we will also quantitatively investigate the biological signaling pathways of platelet contraction using pharmacological agents and platelet agonists using our system. Pharmacologic agents including glycoprotein IIb/IIIa (integrin αIIbβ3) antagonists, Rho kinase inhibitors, calcium inhibitors, and myosin inhibitors, will be used to measure the quantitative effect each biological component has on platelet contraction. In addition, soluble agonists known to activate platelets including thrombin, ADP, thromboxane A2, and epinephrine will be investigated quantitatively and systematically to measure their interactive and synergistic effects on platelet contraction. Furthermore, this device represents a new platform which could be used in drug discovery and to test for changes in platelet contraction with differing pharmacological doses.No relevant conflicts of interest to declare.

At sites of vascular injury, activated platelets exhibit dramatic morphological changes and granule secretion to facilitate recruitment of other platelets and clot formation. In our previous work (Kita et al., 2011), we quantified the effect of the microenvironmental geometry on platelet adhesion using microcontact printing and showed that platelet adhesion and spreading is spatially regulated with microscale resolution. Here we demonstrate that platelet secretion of alpha granules, as indicated with P-selectin staining (a marker for a-granules), is also spatially regulated at the micro/nanoscale. Specifically, we show that fibrinogen micropatterns regulate and determine the spatial distribution of P-selectin at the single platelet level. We also show that tubulin, one of the major components of the platelet cytoskeleton, is also rearranged by the nanoscale geometry of the microenvironment.Using microfabrication techniques we previously developed (Kita et al., 2011), patterned polydimethylsiloxane (PDMS) stamps were “inked” with fluorescently-labeled fibrinogen from human plasma. Fibrinogen patterns were then “microstamped” onto glass coverslips and the printed surface was blocked with 1 % BSA. 20 million/ml of washed human platelets were prepared in Tyrode's buffer and incubated onto protein micropatterned surfaces. Immunofluorescence staining was used to visualize P-selectin surface expression and intracellular distribution, and tubulin distribution of adhered platelets. Platelets were also counter-stained with a fluorescent membrane dye or phalloidin for cell detection or cytoskeleton arrangement, respectively. Images were taken via confocal microscopy with a 63x oil immersion objective.When washed platelets are incubated onto the micropatterned fibrinogen surface, they generally undergo activation and lamellipodia formation. On uniform, non-patterned fibrinogen glass surfaces, (Fig 1A), the average area of spread platelets is 24.86 μm2(n=30, standard error ± 2.27). When the fibrinogen micropattern was larger than the platelets' average area (Fig 1B), multiple platelets covered the micropattern and followed the microenvironmental geometry with high fidelity. P-selectin was detected on the entire platelet surface at slightly higher concentrations at the margins. However, when the features of the fibrinogen micropattern decreased to 5 μm in diameter (Fig 1C, D, and Fig2), platelet spreading was not constrained within the micropattern boundaries. Interestingly, the area of the platelets that spread beyond the fibrinogen micropattern exhibited much higher P-selectin expression than the areas atop the fibrinogen micropattern (Fig 2, arrows), indicating that expression and distribution of P-selectin are spatially regulated by the geometry of the fibrinogen substrate at the microscale.In addition, when platelets are incubated onto fibrinogen microstamps with densely spaced “holes” (0.4–1.0 μm in diameter) blocked with BSA, platelets fully spread and span over those holes. However, dense P-selectin expression was co-localized with these holes, indicating that P-selectin expression and a-granule release are spatially regulated by the underlying protein micropattern (Fig 3A). Interestingly, tubulin showed the opposite trend and only localized to areas directly above the fibrinogen micropattern (Fig 3B). These observations suggest that both platelet a-granule distribution/secretion and cytoskeleton arrangement are regulated at the single platelet level with nanoscale resolution (Fig 3C).Using our platelet adhesion/microstamping technique, we demonstrated that platelets regulate the intracellular trafficking, distribution, and secretion of biomolecules at the nanoscale, responding to the geometry of microenvironment. We will continue to investigate how platelets spatially regulate other aspects of their physiology, including calcium signaling and distribution of receptors/ligands on different substrates such as vWF and collagen.No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

At sites of vascular injury, platelets are exposed to multiple agonists that lead to overall activation and platelet plug formation. Signaling pathways induced by these agonists are known to interact with each other. For example, collagen binding to the platelet collagen receptors, α2β1 integrin, CD36, and glycoprotein VI, induces inside-out signaling that ultimately leads to the activation of the glycoprotein IIb/IIIa receptor for fibrinogen on the platelet surface (Nakamura et al, JCB, 1999).An assay capable of tracking the biological effects of multiple different agonists simultaneously within a single platelet would enhance our understanding of how platelets integrate these different signals at the single-platelet level. This assay would have advantages over current clinical assays in that platelet function tests such as platelet aggregometry or the PFA-100 assess only collective behavior of platelet populations without single platelet resolution. Flow cytometry achieves single platelet resolution but cannot monitor the dynamic changes induced by agonists over time. Platelet adhesion assays have the capability to track individual platelets over time via microscopy, but currently cannot simultaneously monitor the different effects of multiple agonists and their potential interactions.To that end, we developed a modified platelet adhesion assay by using microcontact printing to “stamp” distinct micro-to-nanoscale patterns of different platelet agonists and ligands on glass coverslips. Different parts of each platelet are then exposed to different agonists/ligands with our assay and how the different downstream biological signals interact, synergize, and potentially compete can be monitored overall time at the single platelet level via epifluorescence microscopy.Using microfabrication techniques, patterned polydimethylsiloxane (PDMS) stamps were” inked” with two different known platelet agonists/ligands: Collagen type 1 conjugated with FITC (green) and fibrinogen conjugated to Alexa Fluor 594 (red). Using those stamps, protein micropatterns were then “microstamped” and transferred onto the glass surface, creating spatially distinct microprints with two different proteins (Fig. 1). The protein surface was then blocked with 1% BSA.Platelets were isolated and dyed with a fluorescent membrane marker. Platelets were suspended in Tyrode's buffer (20 million platelets per mL) and incubated with the double micro-patterned surface. After incubation, the surface was washed and imaged with epifluorescence microscopy using a 40x objective.For these initial proof-of-concept experiments, we observed that when individual platelets are simultaneously exposed to separate areas of fibrinogen as well as collagen, they exhibit a strong “preference” for the fibrinogen over collagen (Fig. 2). Indeed, 79.1% of platelets were observed to almost completely (>75% of the surface area) migrate to the fibrinogen micropattern from the collagen micropattern (Fig. 3). Few platelets were spread equally between the two protein micropatterns, which suggest that platelets eventually “choose” which agonist to settle before it get fully spread and immobilized. Interestingly, platelets with more than 75 % surface area on collagen (8.96 % of total platelets measured) tended to be smaller in size, have filopodia, and have intense granule staining compared to platelets on fibrinogen, which were more fully spread with broad lamellipodia. These data suggest that the platelets that migrated to the fibrinogen micropattern compared to those that preferred collagen may be physiologically distinct.Conclusions and Ongoing Efforts: These data establish the viability of our system to investigate the integrative effects of different agonists at the single platelet level. This assay will enable the further understanding of how different agonist-induced signaling pathways interact. Ongoing experiments will include other agonists/ligands such as von Willebrand factor and thrombin. Although the current data assesses morphologic changes of platelets, we are focusing our efforts on using high resolution fluorescence microscopy to measure and monitor the spatial dynamics of calcium signaling, protein phosphorylation of relevant signaling pathways, and cytoskeletal rearrangement when different parts of single platelets are exposed to different agonists.No relevant conflicts of interest to declare.

Abstract 2192