Aging and chronic inflammation are independent risk factors for the development of atherothrombosis and cardiovascular disease. We hypothesized that aging-associated inflammation promotes the development of platelet hyperreactivity and increases thrombotic risk during aging. Functional platelet studies in aged-frail adults and old mice demonstrated that their platelets are hyperreactive and form larger thrombi. We identified tumor necrosis factor α (TNF-α) as the key aging-associated proinflammatory cytokine responsible for platelet hyperreactivity. We further showed that platelet hyperreactivity is neutralized by abrogating signaling through TNF-α receptors in vivo in a mouse model of aging. Analysis of the bone marrow compartments showed significant platelet-biased hematopoiesis in old mice reflected by increased megakaryocyte-committed progenitor cells, megakaryocyte ploidy status, and thrombocytosis. Single-cell RNA-sequencing analysis of native mouse megakaryocytes showed significant reprogramming of inflammatory, metabolic, and mitochondrial gene pathways in old mice that appeared to play a significant role in determining platelet hyperreactivity. Platelets from old mice (where TNF-α was endogenously increased) and from young mice exposed to exogenous TNF-α exhibited significant mitochondrial changes characterized by elevated mitochondrial mass and increased oxygen consumption during activation. These mitochondrial changes were mitigated upon TNF-α blockade. Similar increases in platelet mitochondrial mass were seen in platelets from patients with myeloproliferative neoplasms, where TNF-α levels are also increased. Furthermore, metabolomics studies of platelets from young and old mice demonstrated age-dependent metabolic profiles that may differentially poise platelets for activation. Altogether, we present previously unrecognized evidence that TNF-α critically regulates megakaryocytes resident in the bone marrow niche and aging-associated platelet hyperreactivity and thrombosis.

Key Points Identification of a distinct leukocyte recruitment mechanism by platelet thrombi. Leukocyte migration through thrombi is partially mediated by one or more CXCR1/2 ligands, including NAP-2.

Platelets have evolved a highly specialized membrane skeleton that provides stability to the plasma membrane and facilitates adhesion under high shear stress. The cytoskeletal anchorage of glycoprotein (GP) Ib␣ plays an important role in regulating the membrane skeleton. However, its role in regulating membrane stability remains unknown. To investigate this role, we have developed a new mouse model that expresses wild-type human GPIb␣ (hGPIb␣ WT ), or a mutant form of human GPIb␣ that has a selective defect in its ability to bind filamin A and anchor to the membrane skeleton (hGPIb␣ FWPhe568Ala and Trp570Ala substitutions). Our study demonstrates that the link between platelet GPIb and the cytoskeleton does not alter the intrinsic ligand binding function of GPIb␣ or the ability of the receptor to stimulate integrin ␣ IIb ␤ 3 -dependent spreading. However, exposure of hGPIb␣ FW platelets to pathologic shear rate levels (5000 to 40 000 s ؊1 ) leads to the development of unstable membrane tethers, defective platelet adhesion, and loss of membrane integrity, leading to complete disintegration of the platelet cell body. These outcomes suggest that the GPIb␣-filamin A interaction not only regulates the architecture of the membrane skeleton, but also maintains the mechanical stability of the plasma membrane under conditions of high shear. (Blood. 2011;117(9):2718-2727) IntroductionThe cytoskeleton of all eukaryotic cells plays a fundamental role in regulating the mechanical properties of the cells, influencing cell morphology, deformability, mechanotransduction, and migration. With the development of a high pressure closed circulatory system, blood cells, particularly erythrocytes and platelets, have developed highly specialized cytoskeletal networks linked to the plasma membrane to regulate membrane stability in the face of variable shear forces. The importance of interactions between erythrocyte integral membrane proteins and cytoskeletal structural proteins for erythrocyte membrane stability is well established. Diseases including hereditary spherocytosis and elliptocytosis result in hemolytic anemia as a consequence of genetic defects in erythrocyte membrane skeleton proteins including ankyrin, spectrin, and protein 4.1. These defects disrupt the anchorage of the cytoskeletal network to the plasma membrane resulting in decreased membrane deformability and increased membrane fragmentation. These results ultimately lead to loss of membrane surface area, abnormal cell morphology, and reduced cell survival because of sequestration and clearance in the spleen. 1 In platelets, the actin-rich membrane skeleton is linked to the plasma membrane through the interaction between the cytoskeletal actin cross-linking protein, filamin A and the cytoplasmic tail of glycoprotein (GP) Ib␣ (GPIb␣). 2,3 Molecular defects within the GPIb␣, GPIb␤, or GPIX genes result in the rare bleeding disorder Bernard-Soulier syndrome (BSS), which is characterized by giant platelets and thrombocytopenia. A recent study using GPIb␣-null mice has demo...

Gut ischemia is common in critically ill patients, promoting thrombosis and inflammation in distant organs. The mechanisms linking hemodynamic changes in the gut to remote organ thrombosis remain ill-defined. We demonstrate that gut ischemia in the mouse induces a distinct pulmonary thrombotic disorder triggered by neutrophil macroaggregates. These neutrophil aggregates lead to widespread occlusion of pulmonary arteries, veins, and the microvasculature. A similar pulmonary neutrophil-rich thrombotic response occurred in humans with the acute respiratory distress syndrome. Intravital microscopy during gut ischemia-reperfusion injury revealed that rolling neutrophils extract large membrane fragments from remnant dying platelets in multiple organs. These platelet fragments bridge adjacent neutrophils to facilitate macroaggregation. Platelet-specific deletion of cyclophilin D, a mitochondrial regulator of cell necrosis, prevented neutrophil macroaggregation and pulmonary thrombosis. Our studies demonstrate the existence of a distinct pulmonary thrombotic disorder triggered by dying platelets and neutrophil macroaggregates. Therapeutic targeting of platelet death pathways may reduce pulmonary thrombosis in critically ill patients.

Objective— Acquired von Willebrand syndrome is defined by excessive cleavage of the VWF (von Willebrand Factor) and is associated with impaired primary hemostasis and severe bleeding. It often develops when blood is exposed to nonphysiological flow such as in aortic stenosis or mechanical circulatory support. We evaluated the role of laminar, transitional, and turbulent flow on VWF cleavage and the effects on VWF function. Approach and Results— We used a vane rheometer to generate laminar, transitional, and turbulent flow and evaluate the effect of each on VWF cleavage in the presence of ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type-1 motif, member 13). We performed functional assays to evaluate the effect of these flows on VWF structure and function. Computational fluid dynamics was used to estimate the flow fields and forces within the vane rheometer under each flow condition. Turbulent flow is required for excessive cleavage of VWF in an ADAMTS13-dependent manner. The assay was repeated with whole blood, and the turbulent flow had the same effect. Our computational fluid dynamics results show that under turbulent conditions, the Kolmogorov scale approaches the size of VWF. Finally, cleavage of VWF in this study has functional consequences under flow as the resulting VWF has decreased ability to bind platelets and collagen. Conclusions— Turbulent flow mediates VWF cleavage in the presence of ADAMTS13, decreasing the ability of VWF to sustain platelet adhesion. These findings impact the design of mechanical circulatory support devices and are relevant to pathological environments where turbulence is added to circulation.

von Willebrand factor (VWF) is essential for the induction of arterial thrombosis. In this study, we investigated the critical role of platelet VWF in occlusive thrombosis formation at high shear in mice that do not express platelet VWF (Nbeal2−/−). Using in silico modeling, in vitro high-shear microfluidics, and an in vivo Folts model of arterial thrombosis we reproduced the platelet dynamics that occur under pathological flow in a stenosed vessel. Computational fluid dynamics (CFDs) simulated local hemodynamics in a stenosis based on arterial geometries. The model predicted shear rates, time course of platelet adhesion, and time to occlusion. These predictions were validated in vitro and in vivo. Occlusive thrombosis developed in wild-type control mice that had normal levels of plasma VWF and platelet VWF in vitro and in vivo. Occlusive thrombosis did not form in the Nbeal2−/− mice that had normal plasma VWF and an absence of platelet VWF. Occlusive thrombosis was corrected in Nbeal2−/− microfluidic assays by the addition of exogenous normal platelets with VWF. Combining model and experimental data, we demonstrated the necessary requirement of platelet VWF in α-granules in forming an occlusive thrombus under high shear. These results could inspire new pharmacological targets specific to pathological conditions and prevent arterial thrombosis.

Essentials• von Willebrand factor (VWF) function is shear stress dependent.• Platelet accumulation in a microfluidic assay correlates with VWF levels.• The microfluidic assay discriminates type 1 von Willebrand disease from healthy controls.• The microfluidic flow assay detects responses to therapeutic intervention (DDAVP).

Introduction-Pathological flows in patients with severe aortic stenosis are associated with acquired von Willebrand syndrome. This syndrome is characterized by excessive cleavage of von Willebrand factor by its main protease, A Disintegrin and Metalloproteinase with a Thrombospondin Type 1 Motif, Member 13 (ADAMTS13) leading to decreased VWF function and mucocutaneous bleeding. Aortic valve replacement and correction of the flow behavior to physiological levels reverses the syndrome, supporting the association between pathological flow and acquired von Willebrand syndrome. We investigated the effects of shear and elongational rates on von Willebrand factor cleavage in the presence of ADAMTS13. Methods-We identified acquired von Willebrand syndrome in five patients with severe aortic stenosis. Doppler echography values from these patients were used to develop three computational fluid dynamic (CFD) aortic valve models (normal, mild and severe stenosis). Shear, elongational rates and exposure times identified in the CFD simulations were used as parameters for the design of microfluidic devices to test the effects of pathologic shear and elongational rates on the structure and function of von Willebrand factor. Results-The shear rates (0-10,000s À1), elongational rates (0-1000 s À1) and exposure times (1-180 ms) tested in our microfluidic designs mimicked the flow features identified in patients with aortic stenosis. The shear and elongational rates tested in vitro did not lead to excessive cleavage or decreased function of von Willebrand factor in the presence of the protease. Conclusions-High shear and elongational rates in the presence of ADAMTS13 are not sufficient for excessive cleavage of von Willebrand Factor.