Garth W. Tormoen

Microfluidics and Coagulation Biology

The study of blood ex vivo can occur in closed or open systems, with or without flow. Microfluidic devices, which constrain fluids to a small (typically submillimeter) scale, facilitate analysis of platelet function, coagulation biology, cellular biorheology, adhesion dynamics, and pharmacology and, as a result, can be an invaluable tool for clinical diagnostics. An experimental session can accommodate hundreds to thousands of unique clotting, or thrombotic, events. Using microfluidics, thrombotic events can be studied on defined surfaces of biopolymers, matrix proteins, and tissue factor, under constant flow rate or constant pressure drop conditions. Distinct shear rates can be generated on a device using a single perfusion pump. Microfluidics facilitated both the determination of intraluminal thrombus permeability and the discovery that platelet contractility can be activated by a sudden decrease in flow. Microfluidic devices are ideal for multicolor imaging of platelets, fibrin, and phosphatidylserine and provide a human blood analog to mouse injury models. Overall, microfluidic advances offer many opportunities for research, drug testing under relevant hemodynamic conditions, and clinical diagnostics.

S6K1 and mTOR regulate Rac1-driven platelet activation and aggregation

Platelet activation and thrombus formation are under the control of signaling systems that integrate cellular homeostasis with cytoskeletal dynamics. Here, we identify a role for the ribosome protein S6 kinase (S6K1) and its upstream regulator mTOR in the control of platelet activation and aggregate formation under shear flow. Platelet engagement of fibrinogen initiated a signaling cascade that triggered the activation of S6K1 and Rac1. Fibrinogen-induced S6K1 activation was abolished by inhibitors of Src kinases, but not Rac1 inhibitors, demonstrating that S6K1 acts upstream of Rac1. S6K1 and Rac1 interacted in a protein complex with the Rac1 GEF TIAM1 and colocalized with actin at the platelet lamellipodial edge, suggesting that S6K1 and Rac1 work together to drive platelet spreading. Pharmacologic inhibitors of mTOR and S6K1 blocked Rac1 activation and prevented platelet spreading on fibrinogen, but had no effect on Src or FAK kinase activation. mTOR inhibitors dramatically reduced collagen-induced platelet aggregation and promoted the destabilization of platelet aggregates formed under shear flow conditions. Together, these results reveal novel roles for S6K1 and mTOR in the regulation of Rac1 activity and provide insights into the relationship between the pharmacology of the mTOR system and the molecular mechanisms of platelet activation.

Molecular Imaging of Inflammation and Platelet Adhesion in Advanced Atherosclerosis Effects of Antioxidant Therapy With NADPH Oxidase Inhibition

Background—In atherosclerosis, local generation of reactive oxygen species amplifies the inflammatory response and contributes to plaque vulnerability. We used molecular imaging to test whether inhibition of NADPH oxidase with apocynin would reduce endothelial inflammatory activation and endothelial–platelet interactions, thereby interrupting progression to high-risk plaque phenotype. Methods and Results—Mice deficient for both the low-density lipoprotein receptor and Apobec-1 were studied at 30 weeks of age and again after 10 weeks with or without apocynin treatment (10 or 50 mg/kg per day orally). In vivo molecular imaging of vascular cell adhesion molecule-1 (VCAM 1) P-selectin, and platelet glycoprotein-1b&agr; (GPIb&agr;) in the thoracic aorta was performed with targeted contrast-enhanced ultrasound molecular imaging. Arterial elastic modulus and pulse wave transit time were assessed using ultrahigh frequency ultrasound and invasive hemodynamic measurements. Plaque size and composition were assessed by histology. Molecular imaging in nontreated mice detected a 2-fold increase in P-selectin expression, VCAM-1 expression, and platelet adhesion between 30 and 40 weeks of age. Apocynin reduced all of these endothelial events in a dose-dependent fashion (25% and 50% reduction in signal at 40 weeks for low- and high-dose apocynin). Apocynin also decreased aortic elastic modulus and increased the pulse transit time. On histology, apocynin reduced total monocyte accumulation in a dose-dependent manner as well as platelet adhesion, although total plaque area was reduced in only the high-dose apocynin treatment group. Conclusions—Inhibition of NADPH oxidase in advanced atherosclerosis reduces endothelial activation and platelet adhesion, which are likely responsible for the arrest of plaque growth and improvement of vascular mechanical properties.

Molecular Imaging of Activated von Willebrand Factor to Detect High-Risk Atherosclerotic Phenotype

OBJECTIVES We hypothesized that noninvasive molecular imaging of activated von Willebrand factor (vWF) on the vascular endothelium could be used to detect a high-risk atherosclerotic phenotype. BACKGROUND Platelet-endothelial interactions have been linked to increased inflammatory activation and prothrombotic state in atherosclerosis. These interactions are mediated, in part, by platelet glycoprotein (GP) Ibα, suggesting that dysregulated endothelial vWF is a marker for high-risk atherosclerotic disease. METHODS Microbubbles targeted to activated vWF were prepared by surface conjugation of recombinant GPIbα. Flow-chamber studies were used to evaluate attachment of targeted microbubbles to immobile platelet aggregates bearing activated vWF. Contrast-enhanced ultrasound (CEU) molecular imaging of the aorta from mice was performed: 1) ex vivo after focal crush injury and blood perfusion; and 2) in vivo in mice with advanced atherosclerosis produced by deletion of the low-density lipoprotein receptor and ApoBec-1 editing peptide (LDLR(-/-)/ApoBec-1(-/-)). RESULTS In flow-chamber studies, tracer attachment to vWF was >10-fold greater for microbubbles bearing GPIbα compared with control microbubbles (p < 0.01). In the ex vivo aortic injury model, CEU signal enhancement for vWF-targeted microbubbles occurred primarily at the injury site and was 4-fold greater than at noninjured sites (p < 0.05). In LDLR(-/-)/ApoBec-1(-/-) mice, inflammatory cell infiltrates and dense vWF expression on the intact endothelium were seen in regions of severe plaque formation. Scanning electron microscopy demonstrated widespread platelet-endothelial interaction and only few sites of endothelial erosion. On CEU, signal enhancement for vWF-targeted microbubbles was approximately 4-fold greater (p < 0.05) in LDLR(-/-)/ApoBec-1(-/-) compared with wild-type mice. En face aortic microscopy demonstrated regions where platelet adhesion and microbubble attachment colocalized. CONCLUSIONS Molecular imaging using GPIbα as a targeting moiety can detect the presence of activated vWF on the vascular endothelium. This strategy may provide a means to noninvasively detect an advanced prothrombotic and inflammatory phenotype in atherosclerotic disease.

Pull-off forces measured between hexadecanethiol self-assembled monolayers in air using an atomic force microscope: analysis of surface free energy

The pull-off forces were measured between hexadecanethiol monolayers, self-assembled on gold-coated silicon nitride cantilever tip and silicon wafer, using atomic force microscopy (AFM). The blind tip reconstruction technique was used for determination of the curvature of the AFM tip. The measured pull-off force value remained practically unaffected by a variation of the maximum applied load in a range of 5–80 nN. This result suggests that the use of continuum elastic contact mechanics in the analysis of AFM pull-off force measurements is not as straightforward as usually assumed in the literature reports on similar systems. The surface free energy of hexadecanethiol monolayer on a gold film of γ = 24–27 mJ/m2 was calculated based on the pull-off forces (F), measured using R = 60–80 nm radius tips, and next applying the Derjaguin approximation: F = 4πRγ. These γ-values were found to match the surface free energy value calculated from contact angle data and using Lewis acid–base interfacial free energy theory.

The prothrombotic activity of cancer cells in the circulation

The hemostatic system is often subverted in patients with cancer, resulting in life-threatening venous thrombotic events. Despite the multifactorial and complex etiology of cancer-associated thrombosis, changes in the expression and activity of cancer-derived tissue factor (TF) - the principle initiator of the coagulation cascade - are considered key to malignant hypercoagulopathy and to the pathophysiology of thrombosis. However, many of the molecular and cellular mechanisms coupling the hemostatic degeneration to malignancy remain largely uncharacterized. In this review we discuss some of the tumor-intrinsic and tumor-extrinsic mechanisms that may contribute to the prothrombotic state of cancer, and we bring into focus the potential for circulating tumor cells (CTCs) in advancing our understanding of the field. We also summarize the current status of anti-coagulant therapy for the treatment of thrombosis in patients with cancer.

Analysis of atomic force microscope pull-off forces for gold surfaces portraying nanoscale roughness and specific chemical functionality

Pull-off force measurements were carried out between gold-coated atomic force microscope (AFM) cantilever tips modified with self-assembled monolayers (SAMs) of thiols and similar SAMs prepared on gold-coated silicon wafer surfaces in air with a relative humidity level less than 15%. The gold sputtered silicon wafer substrate formed a granular morphology with dimensions of 30-50 nm. The radii of curvatures for the two different cantilever tips used in this study were 32 nm and 53 nm. Pull-off force results were analyzed using the Derjaguin-Muller-Toporov (DMT) contact mechanics theory to determine both surface free energy (γ) for OH, CH3, CO2H and NH2-terminated monolayers and the resultant work of adhesion (W A) between SAMs. The analysis took into account the coarse morphology of the gold coating. It was found that the γ and W A values determined with the AFM technique approached similar thermodynamic parameters as calculated from the Lifshitz-van der Waals/Lewis acid-base interaction theory using advancing contact angles; however, the high ends of the adhesion force distributions significantly exceeded the predicted values. This discrepancy is discussed in terms of multiple contact points experienced by a probe penetrating into the grain structure of the gold coating.

p21 Activated Kinase Signaling Coordinates Glycoprotein Receptor VI–Mediated Platelet Aggregation, Lamellipodia Formation, and Aggregate Stability Under Shear

Objective—Rho GTPase proteins play a central role in regulating the dynamics of the platelet actin cytoskeleton. Yet, little is known regarding how Rho GTPase activation coordinates platelet activation and function. In this study, we aimed to characterize the role of the Rho GTPase effector, p21 activated kinase (PAK), in platelet activation, lamellipodia formation, and aggregate formation under shear. Approach and Results—Stimulation of platelets with the glycoprotein receptor VI agonist, collagen-related peptide, rapidly activated PAK in a time course preceding phosphorylation of PAK substrates, LIM domain kinase LIMK1 and the MAPK/ERK kinase MEK, and the subsequent activation of MAPKs and Akt. Pharmacological inhibitors of PAK blocked signaling events downstream of PAK and prevented platelet secretion as well as platelet aggregation in response to collagen-related peptide. PAK inhibitors also prevented PAK activation and platelet spreading on collagen surfaces. PAK was also required for the formation of platelet aggregates and to maintain aggregate stability under physiological shear flow conditions. Conclusions—These results suggest that PAK serves as an orchestrator of platelet functional responses after activation downstream of the platelet collagen receptor, glycoprotein receptor VI.

A distribution of AFM pull-off forces for glass microspheres on a symmetrically structured rough surface

The adhesional contact between a particle and a substrate is a fundamental parameter for analyzing pull-off force data generated by atomic force microscopy (AFM). Roughness, present at some scale for all real materials, complicates this task by introducing asperity-controlled contact. Roughness also causes pull-off force data scatter, a well-known phenomenon that is usually neglected in analysis of the AFM pull-off results. This paper presents the first systematic study of roughness effect on the pull-off force magnitude and its distribution characteristics. The results indicate that the scatter in the data decreases with increasing diameter of the probe as compared to the dimension of surface irregularities, but the magnitude of the pull-off force is more severely altered by roughness. The results also show that when particle size is at the same scale as surface roughness, multiple contact points may be made yielding increased adhesion.

Exogenous modification of platelet membranes with the omega-3 fatty acids EPA and DHA reduces platelet procoagulant activity and thrombus formation.

Several studies have implicated the omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in inhibition of normal platelet function, suggesting a role for platelets in EPA- and DHA-mediated cardioprotection. However, it is unclear whether the cardioprotective mechanisms arise from alterations to platelet-platelet, platelet-matrix, or platelet-coagulation factor interactions. Our previous results led us to hypothesize that EPA and DHA alter the ability of platelets to catalyze the generation of thrombin. We tested this hypothesis by exogenously modifying platelet membranes with EPA and DHA, which resulted in compositional changes analogous to increased dietary EPA and DHA intake. Platelets treated with EPA and DHA showed reductions in the rate of thrombin generation and exposure of platelet phosphatidylserine. In addition, treatment of platelets with EPA and DHA decreased thrombus formation and altered the processing of thrombin precursor proteins. Furthermore, treatment of whole blood with EPA and DHA resulted in increased occlusion time and a sharply reduced accumulation of fibrin under flow conditions. These results demonstrate that EPA and DHA inhibit, but do not eliminate, the ability of platelets to catalyze thrombin generation in vitro. The ability of EPA and DHA to reduce the procoagulant function of platelets provides a possible mechanism behind the cardioprotective phenotype in individuals consuming high levels of EPA and DHA.