Engineering vascularized skin-mimetic phantom for non-invasive Raman spectroscopy.

Recent advances in Raman spectroscopy have shown great potential for non-invasive analyte sensing, but the lack of a standardized optical phantom for these measurements has hindered further progress. While many research groups have developed optical phantoms that mimic bulk optical absorption and scattering, these materials typically have strong Raman scattering, making it difficult to distinguish metabolite signals. As a result, solid tissue phantoms for spectroscopy have been limited to highly scattering tissues such as bones and calcifications, and metabolite sensing has been primarily performed using liquid tissue phantoms. To address this issue, we have developed a layered skin-mimetic phantom that can support metabolite sensing through Raman spectroscopy. Our approach incorporates millifluidic vasculature that mimics blood vessels to allow for diffusion akin to metabolite diffusion in the skin. Furthermore, our skin phantoms are mechanically mimetic, providing an ideal model for development of minimally invasive optical techniques. By providing a standardized platform for measuring metabolites, our approach has the potential to facilitate critical developments in spectroscopic techniques and improve our understanding of metabolite dynamics in vivo.

Platelet-primed interactions of coagulation and anticoagulation pathways in flow-dependent thrombus formation.

In haemostasis and thrombosis, platelet, coagulation and anticoagulation pathways act together to produce fibrin-containing thrombi. We developed a microspot-based technique, in which we assessed platelet adhesion, platelet activation, thrombus structure and fibrin clot formation in real time using flowing whole blood. Microspots were made from distinct platelet-adhesive surfaces in the absence or presence of tissue factor, thrombomodulin or activated protein C. Kinetics of platelet activation, thrombus structure and fibrin formation were assessed by fluorescence microscopy. This work revealed: (1) a priming role of platelet adhesion in thrombus contraction and subsequent fibrin formation; (2) a surface-independent role of tissue factor, independent of the shear rate; (3) a mechanism of tissue factor-enhanced activation of the intrinsic coagulation pathway; (4) a local, suppressive role of the anticoagulant thrombomodulin/protein C pathway under flow. Multiparameter analysis using blood samples from patients with (anti)coagulation disorders indicated characteristic defects in thrombus formation, in cases of factor V, XI or XII deficiency; and in contrast, thrombogenic effects in patients with factor V-Leiden. Taken together, this integrative phenotyping approach of platelet-fibrin thrombus formation has revealed interaction mechanisms of platelet-primed key haemostatic pathways with alterations in patients with (anti)coagulation defects. It can help as an important functional add-on whole-blood phenotyping.

Comparative Analysis of Microfluidics Thrombus Formation in Multiple Genetically Modified Mice: Link to Thrombosis and Hemostasis.

Genetically modified mice are indispensable for establishing the roles of platelets in arterial thrombosis and hemostasis. Microfluidics assays using anticoagulated whole blood are commonly used as integrative proxy tests for platelet function in mice. In the present study, we quantified the changes in collagen-dependent thrombus formation for 38 different strains of (genetically) modified mice, all measured with the same microfluidics chamber. The mice included were deficient in platelet receptors, protein kinases or phosphatases, small GTPases or other signaling or scaffold proteins. By standardized re-analysis of high-resolution microscopic images, detailed information was obtained on altered platelet adhesion, aggregation and/or activation. For a subset of 11 mouse strains, these platelet functions were further evaluated in rhodocytin- and laminin-dependent thrombus formation, thus allowing a comparison of glycoprotein VI (GPVI), C-type lectin-like receptor 2 (CLEC2) and integrin α6ß1 pathways. High homogeneity was found between wild-type mice datasets concerning adhesion and aggregation parameters. Quantitative comparison for the 38 modified mouse strains resulted in a matrix visualizing the impact of the respective (genetic) deficiency on thrombus formation with detailed insight into the type and extent of altered thrombus signatures. Network analysis revealed strong clusters of genes involved in GPVI signaling and Ca2+ homeostasis. The majority of mice demonstrating an antithrombotic phenotype in vivo displayed with a larger or smaller reduction in multi-parameter analysis of collagen-dependent thrombus formation in vitro. Remarkably, in only approximately half of the mouse strains that displayed reduced arterial thrombosis in vivo, this was accompanied by impaired hemostasis. This was also reflected by comparing in vitro thrombus formation (by microfluidics) with alterations in in vivo bleeding time. In conclusion, the presently developed multi-parameter analysis of thrombus formation using microfluidics can be used to: (i) determine the severity of platelet abnormalities; (ii) distinguish between altered platelet adhesion, aggregation and activation; and (iii) elucidate both collagen and non-collagen dependent alterations of thrombus formation. This approach may thereby aid in the better understanding and better assessment of genetic variation that affect in vivo arterial thrombosis and hemostasis.

High-throughput elucidation of thrombus formation reveals sources of platelet function variability.

In combination with microspotting, whole-blood microfluidics can provide high-throughput information on multiple platelet functions in thrombus formation. Based on assessment of the inter- and intra-subject variability in parameters of microspot-based thrombus formation, we aimed to determine the platelet factors contributing to this variation. Blood samples from 94 genotyped healthy subjects were analyzed for conventional platelet phenotyping: i.e. hematologic parameters, platelet glycoprotein (GP) expression levels and activation markers (24 parameters). Furthermore, platelets were activated by ADP, CRP-XL or TRAP. Parallel samples were investigated for whole-blood thrombus formation (6 microspots, providing 48 parameters of adhesion, aggregation and activation). Microspots triggered platelet activation through GP Ib-V-IX, GPVI, CLEC-2 and integrins. For most thrombus parameters, inter-subject variation was 2-4 times higher than the intra-subject variation. Principal component analyses indicated coherence between the majority of parameters for the GPVI-dependent microspots, partly linked to hematologic parameters, and glycoprotein expression levels. Prediction models identified parameters per microspot that were linked to variation in agonist-induced αIIbß3 activation and secretion. Common sequence variation of GP6 and FCER1G, associated with GPVI-induced αIIbß3 activation and secretion, affected parameters of GPVI-and CLEC-2-dependent thrombus formation. Subsequent analysis of blood samples from patients with Glanzmann thrombasthenia or storage pool disease revealed thrombus signatures of aggregation-dependent parameters that were subject-dependent, but not linked to GPVI activity. Taken together, this high-throughput elucidation of thrombus formation revealed patterns of inter-subject differences in platelet function, which were partly related to GPVI-induced activation and common genetic variance linked to GPVI, but also included a distinct platelet aggregation component.

Platelet Control of Fibrin Distribution and Microelasticity in Thrombus Formation Under Flow.

OBJECTIVE: Platelet- and fibrin-dependent thrombus formation is regulated by blood flow and exposure of collagen and tissue factor. However, interactions between these blood-borne and vascular components are not well understood. APPROACH AND RESULTS: Here, we developed a method to assess whole-blood thrombus formation on microspots with defined amounts of collagen and tissue factor, allowing determination of the mechanical properties and intrathrombus composition. Confining the collagen content resulted in diminished platelet deposition and fibrin formation at high shear flow conditions, but this effect was compensated by a larger thrombus size and increased accumulation of fibrin in the luminal regions of the thrombi at the expense of the base regions. These thrombi were more dependent on tissue factor-triggered thrombin generation. Microforce nanoindentation analysis revealed a significantly increased microelasticity of thrombi with luminal-oriented fibrin. At a low shear rate, fibrin fibers tended to luminally cover the thrombi, again resulting in a higher microelasticity. Studies with blood from patients with distinct hemostatic insufficiencies indicated an impairment in the formation of a platelet-fibrin thrombus in the cases of dilutional coagulopathy, thrombocytopenia, Scott syndrome, and hemophilia B. CONCLUSIONS: Taken together, our data indicate that (1) thrombin increases the platelet thrombus volume; (2) tissue factor drives the formation of fibrin outside of the platelet thrombus; (3) limitation of platelet adhesion redirects fibrin from bottom to top of the thrombus; (4) a lower shear rate promotes thrombus coverage with fibrin; (5) the fibrin distribution pattern determines thrombus microelasticity; and (6) the thrombus-forming process is reduced in patients with diverse hemostatic defects.

Normal platelet activation profile in patients with peripheral arterial disease on aspirin.

BACKGROUND: Peripheral arterial disease (PAD) is a progressive vascular disease associated with a high risk of cardiovascular morbidity and death. Antithrombotic prevention is usually applied by prescribing the antiplatelet agent aspirin. However, in patients with PAD aspirin fails to provide protection against myocardial infarction and death, only reducing the risk of ischemic stroke. Platelets may play a role in disease development, but this has not been tested by proper mechanistic studies. In the present study, we performed a systematic evaluation of platelet reactivity in whole blood from patients with PAD using two high-throughput assays, i.e. multi-agonist testing of platelet activation by flow cytometry and multi-parameter testing of thrombus formation on spotted microarrays. METHODS: Blood was obtained from 40 patients (38 on aspirin) with PAD in majority class IIa/IIb and from 40 age-matched control subjects. Whole-blood flow cytometry and multiparameter thrombus formation under high-shear flow conditions were determined using recently developed and validated assays. RESULTS: Flow cytometry of whole blood samples from aspirin-treated patients demonstrated unchanged high platelet responsiveness towards ADP, slightly elevated responsiveness after glycoprotein VI stimulation, and decreased responsiveness after PAR1 thrombin receptor stimulation, compared to the control subjects. Most parameters of thrombus formation under flow were similarly high for the patient and control groups. However, in vitro aspirin treatment caused a marked reduction in thrombus formation, especially on collagen surfaces. When compared per subject, markers of ADP- and collagen-induced integrin activation (flow cytometry) strongly correlated with parameters of collagen-dependent thrombus formation under flow, indicative of a common, subject-dependent regulation of both processes. CONCLUSION: Despite of the use of aspirin, most platelet activation properties were in the normal range in whole-blood from class II PAD patients. These data underline the need for more effective antithrombotic pharmacoprotection in PAD.

Insights into platelet-based control of coagulation.

The coagulation process is activated by tight control mechanisms, in which platelets play prominent and unique roles. In thrombosis and hemostasis, activated platelets regulate the coagulation system in various ways: by exposing a phosphatidylserine surface for thrombin formation, by supporting fibrin formation, and by regulating the retraction of a fibrin clot. In this review we discuss the involvement of platelet receptors, other membrane proteins, downstream signaling proteins, cytoskeleton-linked proteins and plasma proteins in these procoagulant functions. Studies with both genetically modified mice and pharmacological inhibitors indicate that, for collagen-adhered platelets, in part common signaling pathways lead to phosphatidylserine exposure, generation of thrombin and fibrin, and retraction of the fibrin clot. However, prolonged Ca(2+) elevation leads to thrombin generation, whereas integrin-dependent signaling stimulates fibrin clot retraction. Contact-dependent signaling pathways, triggered by homotypic platelet-platelet interactions, act in particular via the integrin route.

Femtomolar DNA detection by parallel colorimetric darkfield microscopy of functionalized gold nanoparticles.

We introduce a sensing platform for specific detection of DNA based on the formation of gold nanoparticles dimers on a surface. The specific coupling of a second gold nanoparticle to a surface bound nanoparticle by DNA hybridization results in a red shift of the nanoparticle plasmon peak. This shift can be detected as a color change in the darkfield image of the gold nanoparticles. Parallel detection of hundreds of gold nanoparticles with a calibrated true color camera enabled us to detect specific binding of target DNA. This enables a limit of detection below 1.0×10(-14) M without the need for a spectrometer or a scanning stage.