Chris Ian Jones

Measuring platelet function is important for patient stratification to judge bleeding vs. thrombotic risk and for research into antiplatelet drugs to prevent cardiovascular disease. Variability in platelet function is not fully understood, and large studies of inter-individual variation are making gradual progress using laboratory measurements, but rapid and high-performance hematological tests are also needed. We present here a novel microfluidic technology for platelet function analysis that images light scatter by platelets, using a low-cost, open-source, high-throughput and customizable darkfield imaging system called Dark-Pi. The hardware consists of a camera and a simple LED light source controlled by a Raspberry Pi, with 3D-printed parts. Using the Dark-Pi, platelet aggregation was imaged within adenosine 5′-diphosphate-loaded microcapillaries, revealing clearly visible patterns. This darkfield cellular light scatter approach was previously developed for bacterial cells, and here we adapted and optimized it for directly monitoring platelet aggregation. Capturing high-quality time-resolved images of platelets undergoing activation within microcapillaries allowed us to measure changes in light scattering in platelet-rich plasma that correspond with aggregation measured using conventional laboratory methods. This novel prototype system shows that this approach may have potential for use in large-scale studies of platelet function, combining simplicity with low-cost components and using a disposable dip-and-test microfluidic format.

Platelets are central mediators of haemostasis, responding rapidly to vascular injury through tightly regulated activation and inhibitory mechanisms. This response is mediated by a range of agonists, membrane receptors, and associated signalling pathways, enabling platelets to respond rapidly and specifically to stimuli. This chapter outlines the processes of platelet activation following vascular damage, highlighting the roles of collagen, von Willebrand factor, and secondary agonists such as ADP, thromboxane A₂, and thrombin, alongside endothelial-derived inhibitory signals that restrain excessive activation. It focuses on tyrosine kinase–linked receptors, detailing immunoreceptor tyrosine-based activatory (ITAM and hemITAM) and inhibitory (ITIM/ITSM) pathways, with emphasis on key kinases including Src family kinases, Syk, and Tec family members, and receptors such as GPVI, FcγRIIA, CLEC-2, PECAM-1, and G6b-B. Adhesion receptors, particularly integrins αIIbβ3 and α2β1 and the GPIb- IX-V complex, are examined with respect to inside-out and outside-in signalling and mechanotransduction. Finally, the G-protein-coupled receptors that amplify or inhibit platelet responses are discussed, including PARs, purinergic receptors, thromboxane, and prostaglandin receptors, along with emerging concepts in the regulation of platelets by GPCRs and in therapeutic targeting. The activatory and inhibitory pathways outlined here work together to maintain a balance between haemostasis and thrombosis. This enables a rapid and coordinated platelet response to vascular damage while preventing inappropriate and excessive activation. This maintains vascular integrity while preventing excessive bleeding and pathological thrombosis.

The microfluidic measurement of capillary flow can be used to evaluate the response of biological samples to stimulation, where distance and velocity are altered. Melt-extruded multi-bored microfluidic capillaries allow for high-throughput testing with low device cost, but simple devices may limit control over sample flow when compared to the more complex “lab-on-a-chip” devices produced using advanced microfluidic fabrication methods. Previously, we measured the dynamics of global haemostasis stimulated by thrombin by dipping straight vertical microcapillaries into blood, but only the most rapid response could be monitored, as flow slowed significantly within 30 s. Here, we show an innovative method to extend both the stimulation process and flow measurement time without increasing the cost of the device by adding simple loops to the flexible extruded device. The loops enable longer time-scale measurements by increasing resistance to flow, thereby reducing the dependence on high stimulus concentrations for rapid reactions. The instantaneous velocity and equilibrium heights of straight and looped vertical microcapillary films were assessed with water, plasma and whole blood, showing that the loops create additional frictional resistances, reduce flow velocity and prolong residence times for increased time scales of the stimulation process. A modified pressure balance model was used to capture flow dynamics with the added loop. Looped devices loaded with thrombin and collagen showed an improved detection of blood stimulation responses even with lower stimulus concentrations, compared to straight vertical capillaries. Thrombin-activated blood samples in straight capillaries provided a maximum measurement zone of only 4 mm, while the looped design significantly increased this to 11 mm for much longer time scale measurements. Our results suggest that extending stimulation times can be achieved without complex microfluidic fabrication methods, potentially improving concentration–response blood stimulation assays, and may enhance the accuracy and reliability. We conclude adding a loop to low-cost extruded microfluidic devices may bring microfluidic devices closer to delivering on their promise of widespread, decentralized low-cost evaluation of blood response to stimulation in both research and clinical settings.

The endothelial regulation of platelet activity is incompletely understood. Here we describe novel approaches to find molecular pathways implicated on the platelet–endothelium interaction. Using high‐shear whole‐blood microfluidics, employing coagulant or non‐coagulant conditions at physiological temperature, we observed that the presence of human umbilical vein endothelial cells (HUVEC) strongly suppressed platelet adhesion and activation, via the collagen receptor glycoprotein VI (GPVI) and the PAR receptors for thrombin. Real‐time monitoring of the cytosolic Ca ²⁺ rises in the platelets indicated no major improvement of inhibition by prostacyclin or nitric oxide. Similarly under stasis, exposure of isolated platelets to HUVEC reduced the Ca ²⁺ responses by collagen‐related peptide (CRP‐XL, GPVI agonist) and thrombin (PAR agonist). We then analyzed the label‐free phosphoproteome of platelets (three donors), exposed to HUVEC, CRP‐XL, and/or thrombin. High‐resolution mass spectrometry gave 5463 phosphopeptides, corresponding to 1472 proteins, with good correlation between biological and technical replicates ( R > .86). Stringent filtering steps revealed 26 regulatory pathways (Reactome) and 143 regulated kinase substrates (PhosphoSitePlus), giving a set of protein phosphorylation sites that was differentially (44) or similarly (110) regulated by HUVEC or agonist exposure. The differential regulation was confirmed by stable‐isotope analysis of platelets from two additional donors. Substrate analysis indicated major roles of poorly studied protein kinase classes (MAPK, CDK, DYRK, STK, PKC members). Collectively, these results reveal a resetting of the protein phosphorylation profile in platelets exposed to endothelium or to conventional agonists and to endothelium‐promoted activity of a multi‐kinase network, beyond classical prostacyclin and nitric oxide actors, that may contribute to platelet inhibition.

High throughput, automated, time-lapse imaging for multiparameter haemostasis.

Proteoglycans form a heterogeneous family of proteins with covalently bound sulfated glycosaminoglycans. The extracellular matrix proteoglycan perlecan has been proposed to bind to the platelet- and megakaryocyte-specific receptor G6bB, co-regulating platelet glycoprotein VI (GPVI) signaling. The derived non-sulfate proteoglycan endorepellin was previously shown to enhance platelet adhesion via the collagen receptor, integrin α2β1. Here, we compared the roles of perlecan and other matrix proteoglycans in platelet responses and thrombus formation. We used multi-color flow cytometry to measure the degranulation and integrin αIIbβ3 activation of washed platelets in response to various proteoglycans and collagen-related peptide (CRP), the GPVI agonist. Perlecan, but not endorepellin, enhanced the CRP-induced activation of platelets in a time- and concentration-dependent manner. Similar to collagen, immobilized perlecan, but not other proteoglycans, supported static platelet adhesion and spreading. In-flowed whole-blood perlecan diminished shear-dependent platelet adhesion, while it enforced GPVI-dependent thrombus formation—to a larger extent than endorepellin—to induce more contracted aggregates of activated platelets. We concluded that the sulfated proteoglycan perlecan enhances GPVI-dependent platelet responses extending to thrombus formation, but it does so at the expense of reduced adhesion of platelets under flow.

Platelet and coagulation activation are highly reciprocal processes driven by multi-molecular interactions. Activated platelets secrete several coagulation factors and expose phosphatidylserine, which supports the activation of coagulation factor proteins. On the other hand, the coagulation cascade generates known ligands for platelet receptors, such as thrombin and fibrin. Coagulation factor (F)Xa, (F)XIIIa and activated protein C (APC) can also bind to platelets, but the functional consequences are unclear. Here, we investigated the effects of the activated (anti)coagulation factors on platelets, other than thrombin. Multicolor flow cytometry and aggregation experiments revealed that the ‘supernatant of (hirudin-treated) coagulated plasma’ (SCP) enhanced CRP-XL-induced platelet responses, i.e., integrin α_IIbβ₃ activation, P-selectin exposure and aggregate formation. We demonstrated that FXIIIa in combination with APC enhanced platelet activation in solution, and separately immobilized FXIIIa and APC resulted in platelet spreading. Platelet activation by FXIIIa was inhibited by molecular blockade of glycoprotein VI (GPVI) or Syk kinase. In contrast, platelet spreading on immobilized APC was inhibited by PAR1 blockade. Immobilized, but not soluble, FXIIIa and APC also enhanced in vitro adhesion and aggregation under flow. In conclusion, in coagulation, factors other than thrombin or fibrin can induce platelet activation via GPVI and PAR receptors.