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.

A synthesis approach of mouse studies to identify genes and proteins in arterial thrombosis and bleeding.

Antithrombotic therapies reduce cardiovascular diseases by preventing arterial thrombosis and thromboembolism, but at expense of increased bleeding risks. Arterial thrombosis studies using genetically modified mice have been invaluable for identification of new molecular targets. Because of low sample sizes and heterogeneity in approaches or methodologies, a formal meta-analysis to compare studies of mice with single-gene defects encountered major limitations. To overcome these, we developed a novel synthesis approach to quantitatively scale 1514 published studies of arterial thrombus formation (in vivo and in vitro), thromboembolism, and tail-bleeding of genetically modified mice. Using a newly defined consistency parameter (CP), indicating the strength of published data, comparisons were made of 431 mouse genes, of which 17 consistently contributed to thrombus formation without affecting hemostasis. Ranking analysis indicated high correlations between collagen-dependent thrombosis models in vivo (FeCl3 injury or ligation/compression) and in vitro. Integration of scores and CP values resulted in a network of protein interactions in thrombosis and hemostasis (PITH), which was combined with databases of genetically linked human bleeding and thrombotic disorders. The network contained 2946 nodes linked to modifying genes of thrombus formation, mostly with expression in megakaryocytes. Reactome pathway analysis and network characteristics revealed multiple novel genes with potential contribution to thrombosis/hemostasis. Studies with additional knockout mice revealed that 4 of 8 (Apoe, Fpr2, Ifnar1, Vps13a) new genes were modifying in thrombus formation. The PITH network further: (i) revealed a high similarity of murine and human hemostatic and thrombotic processes and (ii) identified multiple new candidate proteins regulating these processes.

Variable impairment of platelet functions in patients with severe, genetically linked immune deficiencies.

In patients with dysfunctions of the Ca2+ channel ORAI1, stromal interaction molecule 1 (STIM1) or integrin-regulating kindlin-3 (FERMT3), severe immunodeficiency is frequently linked to abnormal platelet activity. In this paper, we studied platelet responsiveness by multiparameter assessment of whole blood thrombus formation under high-shear flow conditions in 9 patients, including relatives, with confirmed rare genetic mutations of ORAI1, STIM1 or FERMT3. In platelets isolated from 5 out of 6 patients with ORAI1 or STIM1 mutations, store-operated Ca2+ entry (SOCE) was either completely or partially defective compared to control platelets. Parameters of platelet adhesion and aggregation on collagen microspots were impaired for 4 out of 6 patients, in part related to a low platelet count. For 4 patients, platelet adhesion/aggregation and procoagulant activity on von Willebrand Factor (VWF)/rhodocytin and VWF/fibrinogen microspots were impaired independently of platelet count, and were partly correlated with SOCE deficiency. Measurement of thrombus formation at low shear rate confirmed a greater impairment of platelet functionality in the ORAI1 patients than in the STIM1 patient. For 3 patients/relatives with a FERMT3 mutation, all parameters of thrombus formation were strongly reduced regardless of the microspot. Bone marrow transplantation, required by 2 patients, resulted in overall improvement of platelet function. We concluded that multiparameter assessment of whole blood thrombus formation in a surface-dependent way can detect: i) additive effects of low platelet count and impaired platelet functionality; ii) aberrant ORAI1-mediated Ca2+ entry; iii) differences in platelet activation between patients carrying the same ORAI1 mutation; iv) severe platelet function impairment linked to a FERMT3 mutation and bleeding history.

Gradual increase in thrombogenicity of juvenile platelets formed upon offset of prasugrel medication.

In patients with acute coronary syndrome, dual antiplatelet therapy with aspirin and a P2Y12 inhibitor like prasugrel is prescribed for one year. Here, we investigated how the hemostatic function of platelets recovers after discontinuation of prasugrel treatment. Therefore, 16 patients who suffered from ST-elevation myocardial infarction were investigated. Patients were treated with aspirin (100 mg/day, long-term) and stopped taking prasugrel (10 mg/day) after one year. Blood was collected at the last day of prasugrel intake and at 1, 2, 5, 12 and 30 days later. Platelet function in response to ADP was normalized between five and 30 days after treatment cessation and in vitro addition of the reversible P2Y12 receptor antagonist ticagrelor fully suppressed the regained activation response. Discontinuation of prasugrel resulted in the formation of an emerging subpopulation of ADP-responsive platelets, exhibiting high expression of active integrin αIIbß3. Two different mRNA probes, thiazole orange and the novel 5'Cy5-oligo-dT probe revealed that this subpopulation consisted of juvenile platelets, which progressively contributed to platelet aggregation and thrombus formation under flow. During offset, juvenile platelets were overall more reactive than older platelets. Interestingly, the responsiveness of both juvenile and older platelets increased in time, pointing towards a residual inhibitory effect of prasugrel on the megakaryocyte level. In conclusion, the gradual increase in thrombogenicity after cessation of prasugrel treatment is due to the increased activity of juvenile platelets.

Identification of platelet function defects by multi-parameter assessment of thrombus formation.

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>α6ß1, αIIbß3>α2ß1>CD36, α5ß1, αvß3. 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.

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.

Supporting roles of platelet thrombospondin-1 and CD36 in thrombus formation on collagen.

OBJECTIVE: Platelets abundantly express the membrane receptor CD36 and store its ligand thrombospondin-1 (TSP1) in the α-granules. We investigated whether released TSP1 can support platelet adhesion and thrombus formation via interaction with CD36. APPROACH AND RESULTS: Mouse platelets deficient in CD36 showed reduced adhesion to TSP1 and subsequent phosphatidylserine expression. Deficiency in either CD36 or TSP1 resulted in markedly increased dissolution of thrombi formed on collagen, although thrombus buildup was unchanged. In mesenteric vessels in vivo, deficiency in CD36 prolonged the time to occlusion and enhanced embolization, which was in agreement with earlier observations in TSP1-deficient mice. Thrombi formed using wild-type blood stained positively for secreted TSP1. Releasate from wild-type but not from TSP1-deficient platelets enhanced platelet activation, phosphatidylserine expression, and thrombus formation on collagen. The enhancement was dependent on CD36 because it was without effect on thrombus formation by CD36-deficient platelets. CONCLUSIONS: These results demonstrate an anchoring role of platelet-released TSP1 via CD36 in platelet adhesion and collagen-dependent thrombus stabilization. Thus, the TSP1-CD36 tandem is another platelet ligand-receptor axis contributing to the maintenance of a stable thrombus.

Distinct role of von Willebrand factor triplet bands in glycoprotein Ib-dependent platelet adhesion and thrombus formation under flow.

Multimeric glycoprotein von Willebrand factor (VWF) exhibits a unique triplet structure of individual oligomers, resulting from ADAMTS-13 (a disintegrin and metalloproteinase with thrombospondin type 1 motifs 13) cleavage. The faster and slower migrating triplet bands of a given VWF multimer have one shorter or longer N-terminal peptide sequence, respectively. Within this peptide sequence, the A1 domain regulates interaction of VWF with platelet glycoprotein (GP)Ib. Therefore, platelet-adhesive properties of two VWF preparations with similar multimeric distribution but different triplet composition were investigated for differential functional activities. Preparation A was enriched in intermediate triplet bands, whereas preparation B predominantly contained larger triplet bands. Binding studies revealed that preparation A displayed a reduced affinity for recombinant GPIb but an unchanged affinity for collagen type III when compared to preparation B. Under high-shear flow conditions, preparation A was less active in recruiting platelets to collagen type III. Furthermore, when added to blood from patients with von Willebrand disease (VWD), defective thrombus formation was less restored. Thus, VWF forms lacking larger-size triplet bands appear to have a decreased potential to recruit platelets to collagen-bound VWF under arterial flow conditions. By implication, changes in triplet band distribution observed in patients with VWD may result in altered platelet adhesion at high-shear flow.

Atheroprotective effect of dietary walnut intake in ApoE-deficient mice: involvement of lipids and coagulation factors.

INTRODUCTION: Consumption of n-3 polyunsaturated fatty acids (PUFA) and antioxidant polyphenols is considered to decline the risk of cardiovascular disease. MATERIALS AND METHODS: To provide an explanation for this cardioprotective effect, we performed an intervention study with proatherogenic Apoe(-/-) mice which were fed during eight weeks with a high fat diet supplemented with either walnuts (rich in n-3 PUFA and antioxidant compounds), walnut oil (with n-3 PUFA only) or sunflower oil as a control (12 mice per group). RESULTS: Feeding walnuts, but not walnut oil, caused a 55% reduction in atherosclerotic plaque development in the aortic arch in comparison to the control diet. This was associated with reduced staining of plaques for CD36, a scavenger receptor expressed by macrophages. Feeding mice with walnuts also lowered plasma levels of triglycerides, cholesterol and prothrombin with 36%, 23% and 21 %, respectively, compared to control diet. In addition, accumulation of lipids in the liver was decreased, while plasma antioxidant capacity was increased. On the other hand, feeding mice with walnut oil did not provoke significant changes in these parameters in comparison to the control diet. Platelet activation and thrombus formation under flow remained unchanged with either diet. CONCLUSIONS: In Apoe(-/-) mice on high fat diet, intake of dietary walnut (but not walnut oil) beneficially influences lipid metabolism and atherosclerotic plaque development, with no more than limited effects on platelet and coagulation function.

Monitoring in vitro thrombus formation with novel microfluidic devices.

Cardiovascular disease is a major cause of mortality globally and is subject to ongoing research to improve clinical treatment. It is established that activation of platelets and coagulation are central to thrombosis, yet at different extents in the arterial and venous system. In vitro perfusion chamber technology has contributed significant knowledge on the function of platelets in the thrombotic process under shear conditions. Recent efforts to downscale this technique with a variety of microfluidic devices has opened new possibilities to study this process under precisely controlled flow conditions. Such microfluidic devices possess the capability to execute platelet function tests more quickly than current assays, while using small blood samples. Gradually becoming available to the clinic now, they may provide a new means to manage the treatment of cardiovascular diseases, although accurate validation studies still are missing. This review highlights the progress that has been made in monitoring aspects of thrombus formation using microfluidic devices.

Key role of glycoprotein Ib/V/IX and von Willebrand factor in platelet activation-dependent fibrin formation at low shear flow.

A microscopic method was developed to study the role of platelets in fibrin formation. Perfusion of adhered platelets with plasma under coagulating conditions at a low shear rate (250(-1)) resulted in the assembly of a star-like fibrin network at the platelet surface. The focal fibrin formation on platelets was preceded by rises in cytosolic Ca(2+), morphologic changes, and phosphatidylserine exposure. Fibrin formation was slightly affected by α(IIb)ß(3) blockage, but it was greatly delayed and reduced by the following: inhibition of thrombin or platelet activation; interference in the binding of von Willebrand factor (VWF) to glycoprotein Ib/V/IX (GpIb-V-IX); plasma or blood from patients with type 1 von Willebrand disease; and plasma from mice deficient in VWF or the extracellular domain of GpIbα. In this process, the GpIb-binding A1 domain of VWF was similarly effective as full-length VWF. Prestimulation of platelets enhanced the formation of fibrin, which was abrogated by blockage of phosphatidylserine. Together, these results show that, in the presence of thrombin and low shear flow, VWF-induced activation of GpIb-V-IX triggers platelet procoagulant activity and anchorage of a star-like fibrin network. This process can be relevant in hemostasis and the manifestation of von Willebrand disease.