Characterization of Atherosclerotic Plaque Coating for Thrombosis Microfluidics Assays.

INTRODUCTION: Studying arterial thrombus formation by in vitro flow assays is a widely used approach. Incorporating human atherosclerotic plaque material as a thrombogenic surface in these assays represents a method to model the pathophysiological environment of thrombus formation upon plaque disruption. Up until now, achieving a homogeneous coating of plaque material and subsequent reproducible platelet adhesion has been challenging. Here, we characterized a novel method for coating of plaque material on glass coverslips for use in thrombosis microfluidic assays. METHODS: A homogenate of human atherosclerotic plaques was coated on glass coverslips by conventional manual droplet coating or by spin coating. Prior to coating, a subset of coverslips was plasma treated. Water contact angle measurements were performed as an indicator for the hydrophilicity of the coverslips. Homogeneity of plaque coatings was determined using profilometric analysis and scanning electron microscopy. Thrombogenicity of the plaque material was assessed in real time by microscopic imaging while perfusing whole blood at a shear rate of 1500 s-1 over the plaque material. RESULTS: Plasma treatment of glass coverslips, prior to spin coating with plaque material, increased the hydrophilicity of the coverslip compared to no plasma treatment. The most homogeneous plaque coating and highest platelet adhesion was obtained upon plasma treatment followed by spin coating of the plaque material. Manual plaque coating on non-plasma treated coverslips yielded lowest coating homogeneity and platelet adhesion and activation. CONCLUSION: Spin coating of atherosclerotic plaque material on plasma treated coverslips leads to a more homogenous coating and improved platelet adhesion to the plaque when compared to conventional droplet coating on non-plasma treated coverslips. These properties are beneficial in ensuring the quality and reproducibility of flow experiments. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12195-021-00713-9.

Anticoagulant and antithrombotic properties of intracellular protease-activated receptor antagonists.

BACKGROUND: Blockade of the thrombin receptors protease-activated receptor (PAR)1 and PAR4 with pepducins, cell-penetrating lipopeptides based on the third intracellular loop of PAR1 and PAR4, effectively inhibits platelet aggregation. We have previously shown that PAR1 pepducin also exerts an anticoagulant activity by partial inhibition of the thrombin plus collagen-induced externalization of phosphatidylserine (PS) at the platelet plasma membrane. OBJECTIVE: In the present study we examined the effects of PAR1 and PAR4 pepducins on tissue factor (TF)-initiated thrombin generation in platelet-rich plasma (PRP) and the interaction between PAR4 pepducin-loaded mouse platelets and a growing thrombus to confirm the relevance of the in vitro data. RESULTS: Localization of pepducins at the inner leaflet of the plasma membrane was confirmed with a fluorescence resonance energy transfer assay. Both the PAR1 pepducin, P1pal12, and the PAR4 pepducin, P4pal10, inhibited TF-initiated thrombin generation in PRP. Concentrations of P1pal12 and P4pal10, which blocked the thrombin-induced influx of extracellular calcium ions and inhibited platelet aggregation, reduced the rate of thrombin generation during the propagation phase by 38% and 36%, respectively. Whether this anticoagulant effect is relevant in inhibiting in vivo arterial thrombin growth is uncertain because P4pal10 prevented the incorporation of platelets in a growing thrombus. CONCLUSIONS: Our findings suggest that in spite of their potential anticoagulant activities the in vivo antithrombotic effect of intracellular PAR pepducins is mainly based on inhibiting platelet-platelet interactions.

Rolling and adhesion of apoptotic monocytes is impaired by loss of functional cell surface-expressed P-selectin glycoprotein ligand-1.

BACKGROUND: Apoptosis induces cellular membrane changes that are thought to be linked to thrombotic processes, for example, surface exposure of procoagulant phosphatidylserine (PtdSer), upregulation of tissue factor (TF), and microvesicle formation. The latter, though, could downregulate this cellular response by shedding prothrombotic membrane elements, for example, integrins and TF. To test this hypothesis, etoposide-treated, apoptotic, monocytic cells (human monocytic leukemia cell line [THP-1]) were examined for rolling and adhesion on adherent platelets and for TF expression. METHODS AND RESULTS: Etoposide treatment did not result in a significant change in TF antigen expression. However, TF activity, measured in a continuous factor Xa generation assay, was increased fivefold concomitantly with increased exposure of PtdSer. Laminar flow adhesion assays specific for interaction between P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1) revealed that in contrast to non-treated cells, apoptotic cells did not roll or firmly attach on adherent platelets. Lack of apoptotic THP-1 platelet interaction could be attributed to both a loss of cell surface-expressed PSGL-1 and loss of functional PSGL-1 as a result of disruption of the binding of PSGL-1 with the cytoskeleton. CONCLUSION: Etoposide-induced apoptosis in THP-1 cells evokes a procoagulant response by increasing TF activity associated with an increased PtdSer exposure. However, in contrast to TF, PSGL-1 shedding and loss of function, makes that apoptotic monocytes are unlikely involved in a thrombotic action because of their inability to adhere to an injured vessel wall or developing thrombus.

von Willebrand factor stimulates thrombin-induced exposure of procoagulant phospholipids on the surface of fibrin-adherent platelets.

Studies from our laboratory have demonstrated that von Willebrand factor (VWF) stimulates thrombin generation in platelet-rich plasma. The precise role of VWF and fibrin in this reaction, however, remained to be clarified. In the present study we utilized thrombin-free planar fibrin layers and washed platelets to examine the relationship between platelet-fibrin interaction and exposure of coagulation-stimulating phosphatidylserine (PS) under conditions of low and high shear stress. Our study confirms that platelet adhesion to fibrin at a shear rate of 1000 s(-1) requires fibrin-bound VWF. The cytosolic calcium concentration ([Ca(2+)]i) of stationary platelets was not elevated and PS exposing platelets were virtually absent (2 +/- 2%). However, thrombin activation resulted in a marked increase in the number of PS exposing platelets (up to 85 +/- 14%) along with a transient elevation in [Ca(2+)]i from 0.05 micro mol L(-1) up to 1.1 +/- 0.2 micro mol L(-1). Platelet adhesion to fibrin at a shear rate of 50 s(-1) is mediated by thrombin but not by fibrin-bound VWF. The [Ca(2+)]i of these thrombin-activated platelets was elevated (0.2 +/- 0.1 micro mol L(-1)), but only a minority of the platelets (11 +/- 8%) exposed PS. The essential role of VWF in this thrombin-induced procoagulant response became apparent from low shear rate perfusion studies over fibrin that was incubated with VWF and botrocetin. After treatment with thrombin, the majority of the adherent platelets (57 +/- 23%) exposed PS and had peak values of [Ca(2+)]i of about 0.6 micro mol L(-1). Taken together, these results demonstrate that thrombin-induced exposure of PS and high calcium response on fibrin-adherent platelets depends on shear- or botrocetin-induced VWF-platelet interaction.