Assays measuring platelet aggregation (thrombus formation) at arterial shear rate mostly use collagen as only platelet-adhesive surface. Here we report a multi-surface and multi-parameter flow assay to characterize thrombus formation in whole blood from healthy subjects and patients with platelet function deficiencies. A systematic comparison is made of 52 adhesive surfaces with components activating the main platelet-adhesive receptors, and of eight output parameters reflecting distinct stages of thrombus formation. Three types of thrombus formation can be identified with a predicted hierarchy of the following receptors: glycoprotein (GP)VI, C-type lectin-like receptor-2 (CLEC-2)>GPIb>α₆β₁, α_IIbβ₃>α₂β₁>CD36, α₅β₁, α_vβ₃. Application with patient blood reveals distinct abnormalities in thrombus formation in patients with severe combined immune deficiency, Glanzmann’s thrombasthenia, Hermansky–Pudlak syndrome, May–Hegglin anomaly or grey platelet syndrome. We suggest this test may be useful for the diagnosis of patients with suspected bleeding disorders or a pro-thrombotic tendency.

Traditionally, in vitro flow chamber experiments and in vivo arterial thrombosis studies have been proven to be of vital importance to elucidate the mechanisms of platelet thrombus formation after vessel wall injury. In recent years, it has become clear that platelets also act as modulators of inflammatory processes, such as atherosclerosis. A key element herein is the complex crosstalk between platelets, the coagulation system, leukocytes and the activated endothelium. This review provides insight into the platelet-endothelial interface, based on in vitro flow chamber studies and cross referenced with in vivo thrombosis studies. The main mechanisms of platelet interaction with the activated endothelium encompass i) platelet rolling via interaction of platelet glycoprotein Ib-IX-V with endothelial-released von Willebrand factor with a supporting role for the P-selectin - P-selectin glycoprotein ligand 1 axis, followed by ii) firm platelet adhesion to the endothelium via interaction of platelet α_IIbβ₃ with endothelial α_vβ₃ and intercellular adhesion molecule 1, and iii) a stimulatory role for thrombin, the thrombospondin-1 - CD36 axis and cyclooxygenase 1 in subsequent platelet activation and stable thrombus formation. In addition, the molecular mechanisms underlying the stimulatory effect of platelets on leukocyte transendothelial migration, a key mediator of atheroprogression, are discussed. Throughout the review emphasis is placed on recommendations for setting up, reporting, interpreting and comparing endothelial-lined flow chamber studies and on suggestions for future studies.

Background—: Platelets are central to the process of hemostasis, rapidly aggregating at sites of blood vessel injury and acting as coagulation nidus sites. On interaction with the subendothelial matrix, platelets are transformed into balloonlike structures as part of the hemostatic response. It remains unclear, however, how and why platelets generate these structures. We set out to determine the physiological relevance and cellular and molecular mechanisms underlying platelet membrane ballooning. Methods and Results—: Using 4-dimensional live-cell imaging and electron microscopy, we show that human platelets adherent to collagen are transformed into phosphatidylserine-exposing balloonlike structures with expansive macro/microvesiculate contact surfaces, by a process that we termed procoagulant spreading. We reveal that ballooning is mechanistically and structurally distinct from membrane blebbing and involves disruption to the platelet microtubule cytoskeleton and inflation through fluid entry. Unlike blebbing, procoagulant ballooning is irreversible and a consequence of Na ⁺ , Cl ^– , and water entry. Furthermore, membrane ballooning correlated with microparticle generation. Inhibition of Na ⁺ , Cl ^– , or water entry impaired ballooning, procoagulant spreading, and microparticle generation, and it also diminished local thrombin generation. Human Scott syndrome platelets, which lack expression of Ano-6, also showed a marked reduction in membrane ballooning, consistent with a role for chloride entry in the process. Finally, the blockade of water entry by acetazolamide attenuated ballooning in vitro and markedly suppressed thrombus formation in vivo in a mouse model of thrombosis. Conclusions—: Ballooning and procoagulant spreading of platelets are driven by fluid entry into the cells, and are important for the amplification of localized coagulation in thrombosis.

Custom-made and commercial parallel-plate flow chambers are widely used for studies of platelet activation and thrombus formation in whole blood at defined shear rates. When used in a reproducible way, such flow chamber devices give valuable information on the thrombogenic potential of human, mouse, or rat blood. This article aims to provide a practical guide for the use of parallel-plate flow chambers in combination with routine microscopic imaging techniques. The following methodological aspects are addressed: preparation of surface coatings, calculation of blood flow and shear rate, control of pre-analytical variables, protocols for routine performing of flow chamber tests with non-coagulating or coagulating blood, and procedures for real-time and end-point analysis of thrombus formation. Frequently encountered experimental problems and artifacts are discussed, as well as possibilities for using flow chamber devices as a diagnostic tool to test antithrombotic medication.

Objective—: Atherothrombosis is the main cause of myocardial infarction and ischemic stroke. Although the extrinsic (tissue factor–factor VIIa [FVIIa]) pathway is considered as a major trigger of coagulation in atherothrombosis, the role of the intrinsic coagulation pathway via coagulation FXII herein is unknown. Here, we studied the roles of the extrinsic and intrinsic coagulation pathways in thrombus formation on atherosclerotic plaques both in vivo and ex vivo. Approach and Results—: Plaque rupture after ultrasound treatment evoked immediate formation of subocclusive thrombi in the carotid arteries of Apoe ^−/− mice, which became unstable in the presence of structurally different FXIIa inhibitors. In contrast, inhibition of FVIIa reduced thrombus size at a more initial stage without affecting embolization. Genetic deficiency in FXII (human and mouse) or FXI (mouse) reduced ex vivo whole-blood thrombus and fibrin formation on immobilized plaque homogenates. Localization studies by confocal microscopy indicated that FXIIa bound to thrombi and fibrin particularly in luminal-exposed thrombus areas. Conclusions—: The FVIIa- and FXIIa-triggered coagulation pathways have distinct but complementary roles in atherothrombus formation. The tissue factor–FVIIa pathway contributes to initial thrombus buildup, whereas FXIIa bound to thrombi ensures thrombus stability.