Dasatinib inhibits actin fiber reorganization and promotes endothelial cell permeability through RhoA-ROCK pathway.

Treatment with dasatinib, a tyrosine kinase inhibitor, is associated with edema, pleural effusion, and pulmonary edema. We investigated the effect of dasatinib on the barrier function of human microvascular endothelial cells-1 (HMEC-1) in vitro and in vivo. The permeability of HMEC-1 to fluorescein isothiocyante (FITC)-dextran increased in Transwell chambers within 5 min following the addition of therapeutic concentrations of dasatinib. The change in permeability was associated with increased activation of RhoA GTPase and its effector Rho-associated coiled-coil kinase 1(ROCK1). RhoA inhibitor C3 transferase almost completely inhibited dasatinib-induced increase in permeability. Under similar conditions, imatinib had no effect on permeability or activation of RhoA. Since integrin-induced cell spreading suppresses RhoA activation, we examined the effect of dasatinib on cell spreading on fibronectin substrate. Dasatinib impaired endothelial cell spreading in a concentration-dependent manner and induced disorganization of actin fibers. Tyrosine kinases play an essential role in transmitting signals from integrins to RhoA and we examined tyrosine phosphorylation of several cytoskeletal proteins. Dasatinib markedly inhibited tyrosine phosphorylation of p130 Crk-associated substrate (p130cas), paxillin and vinculin. These results suggest that the inhibition of tyrosine phosphorylation of the focal adhesion plaque components by dasatinib may alter the assembly of actin fibers resulting in the activation of RhoA/ROCK pathway. Consistent with these findings, dasatinib-induced increase in the permeability was blocked by ROCK inhibitor y27632. In vivo administration of y27632, significantly inhibited the dasatinib-induced extravasation of Evans blue in mice and dasatinib-induced increase in microvascular permeability was attenuated in ROCK1-deficient mice. These findings suggest that ROCK inhibitors could serve as therapeutic modalities to ameliorate the dasatinib-induced pulmonary changes.

Wdr1-Dependent Actin Reorganization in Platelet Activation.

In resting platelets, the integrin αIIbß3 is present in a low-affinity "bent" state. During platelet aggregation, intracytoplasmic signals induce conformational changes (inside-out signaling) that result in a "swung-out" conformation competent to bind ligands such as fibrinogen. The cytoskeleton plays an essential role in αIIbß3 activation. We investigated the role of the actin interacting protein Wdr1 in αIIbß3 activation. Wdr1-hypomorphic mice had a prolonged bleeding time (> 10 minutes) compared to that of wild-type mice (2.1 ± 0.7 minutes). Their platelets had impaired aggregation to collagen and thrombin. In a FeCl3 induced carotid artery thrombosis model, vessel occlusion in Wdr1-hypomorphic mice was prolonged significantly compared to wild-type mice (9.0 ± 10.5 minutes versus 5.8 ± 12.6 minutes (p = 0.041). Activation-induced binding of JON/A (a conformation-specific antibody to activated αIIbß3) was significantly less in Wdr1-hypomorphic platelets at various concentrations of collagen, indicating impaired inside-out activation of αIIbß3, despite a normal calcium response. Actin turnover, assessed by measuring F-actin and G-actin ratios during collagen- and thrombin-induced platelet aggregation, was highly impaired in Wdr1-hypomorphic platelets. Furthermore, talin failed to redistribute and translocate to the cytoskeleton following activation in Wdr1-hypomorphic platelets. These studies show that Wdr1 is essential for talin-induced activation of αIIbß3 during platelet activation.

Rho associated coiled-coil kinase-1 regulates collagen-induced phosphatidylserine exposure in platelets.

BACKGROUND: The transbilayer movement of phosphatidylserine mediates the platelet procoagulant activity during collagen stimulation. The Rho-associated coiled-coil kinase (ROCK) inhibitor Y-27632 inhibits senescence induced but not activation induced phosphatidylserine exposure. To investigate further the specific mechanisms, we now utilized mice with genetic deletion of the ROCK1 isoform. METHODS AND RESULTS: ROCK1-deficient mouse platelets expose significantly more phosphatidylserine and generate more thrombin upon activation with collagen compared to wild-type platelets. There were no significant defects in platelet shape change, aggregation, or calcium response compared to wild-type platelets. Collagen-stimulated ROCK1-deficient platelets also displayed decreased phosphorylation levels of Lim Kinase-1 and cofilin-1. However, there was no reduction in phosphorylation levels of myosin phosphatase subunit-1 (MYPT1) or myosin light chain (MLC). In an in vivo light/dye-induced endothelial injury/thrombosis model, ROCK1-deficient mice presented a shorter occlusion time in cremasteric venules when compared to wild-type littermates (3.16 ± 1.33 min versus 6.6 ± 2.6 min; p = 0.01). CONCLUSIONS: These studies define ROCK1 as a new regulator for collagen-induced phosphatidylserine exposure in platelets with functional consequences on thrombosis. This effect was downstream of calcium signaling and was mediated by Lim Kinase-1 / cofilin-1-induced cytoskeletal changes.

Developmental endothelial locus-1 (Del-1) mediates clearance of platelet microparticles by the endothelium.

BACKGROUND: Phosphatidylserine-expressing microparticles circulate in blood with a short half-life of <10 minutes. We tested the role of an endothelium-derived phosphatidylserine-binding opsonin, developmental endothelial locus-1 (Del-1), in the uptake of platelet microparticles. METHODS AND RESULTS: Cultured human umbilical vein and microvascular endothelial cells avidly engulf BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene)-maleimide-labeled platelet microparticles. Microparticle uptake was inhibited by a monoclonal antibody to Del-1 (P=0.027) and by annexin A5 (P=0.027), abciximab (P=0.027), a monoclonal antibody to integrin αVß3 (P=0.027), and chlorpromazine (P=0.027). These results suggest that Del-1 mediates phosphatidylserine- and integrin-dependent endothelial uptake of microparticles by endocytosis. To assess the in vivo significance, we infused fluorescent platelet microparticles into the inferior vena cava of mice and harvested endothelial cells from the pulmonary and systemic circulation. Compared with their wild-type littermates, Del-1-deficient mice had decreased uptake in endothelial cells in lung (3.07±1.9 versus 1.09±1.3, P=0.02) and liver (2.85±1.1 versus 1.35±0.92, P=0.01). Furthermore, after endotoxin administration, Del-1-deficient mice displayed an increase in the level of microparticles compared with wild-type mice (P=0.02). CONCLUSIONS: These studies show a physiological role for Del-1 in the clearance of phosphatidylserine-expressing microparticles by endothelium.

Phagocytosis of platelet microvesicles and beta2- glycoprotein I.

The majority of the antiphospholipid antibodies, present in patients with antiphospholipid syndrome, are directed against conformational epitopes in beta2-glycoprotein I. beta2-glycoprotein I is an anionic phospholipid-binding 50-kDa plasma protein whose physiological role is not clear. Here we investigate the role of beta2-glycoprotein I in the phagocytosis of phosphatidylserine-expressing platelet microvesicles and the effect of autoantibodies to beta2-glycoprotein I on this process. We labelled the glycans of beta2-glycoprotein I with BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene)-hydrazide without affecting its phospholipid binding capacity. BODIPY-beta2-glycoprotein I bound to platelet microvesicles in a concentration-dependent manner and promoted the phagocytosis of platelet microvesicles by THP-1 derived macrophages in vitro at physiological plasma concentrations with a half maximal effect at approximately 10 microg/ml. beta2-glycoprotein I-stimulated phagocytosis was inhibited by annexin A5 and the phosphatidylserine-binding C1C2 fragment of lactadherin. Furthermore, immunoaffinity purified beta2-glycoprotein I-dependent antiphospholipid antibodies from five patients with antiphospholipid syndrome inhibited the phagocytosis in a concentration-dependent manner. These studies suggest that the binding of beta2-glycoprotein I to phosphatidylserine-expressing procoagulant platelet microvesicles may promote their clearance by phagocytosis and autoantibodies to beta2-glycoprotein I may inhibit this process to induce a procoagulant state.

Lactadherin and clearance of platelet-derived microvesicles.

The transbilayer movement of phosphatidylserine from the inner to the outer leaflet of the membrane bilayer during platelet activation is associated with the release of procoagulant phosphatidylserine-rich small membrane vesicles called platelet-derived microvesicles. We tested the effect of lactadherin, which promotes the phagocytosis of phosphatidylserine-expressing lymphocytes and red blood cells, in the clearance of platelet microvesicles. Platelet-derived microvesicles were labeled with BODIPY-maleimide and incubated with THP-1-derived macrophages. The extent of phagocytosis was quantified by flow cytometry. Lactadherin promoted phagocytosis in a concentration-dependent manner with a half-maximal effect at approximately 5 ng/mL. Lactadherin-deficient mice had increased number of platelet-derived microvesicles in their plasma compared with their wild-type littermates (950 +/- 165 vs 4760 +/- 650; P = .02) and generated 2-fold more thrombin. In addition, splenic macrophages from lactadherin-deficient mice showed decreased capacity to phagocytose platelet-derived microvesicles. In an in vivo model of light/dye-induced endothelial injury/thrombosis in the cremasteric venules, lactadherin-deficient mice had significantly shorter time for occlusion compared with their wild-type littermate controls (5.93 +/- 0.43 minutes vs 9.80 +/- 1.14 minutes;P = .01). These studies show that lactadherin mediates the clearance of phosphatidylserine-expressing platelet-derived microvesicles from the circulation and that a defective clearance can induce a hypercoagulable state.

Lactadherin mediates sickle cell adhesion to vascular endothelial cells in flowing blood.

Increased exposure of sickle red blood cells to phosphatidylserine promotes its adhesion to the endothelium. A monoclonal antibody to lactadherin, a phosphatidylserine binding protein, inhibits sickle cell adhesion to histamine-stimulated endothelial cells in flowing blood. Added lactadherin enhances the adhesion via the integrin alphaVbeta3. These results indicate that lactadherin can mediate phosphatidylserine-expressing sickle cell adhesion to the endothelium.

Lupus-derived antiprothrombin autoantibodies from a V gene phage display library are specific for the kringle 2 domain of prothrombin.

Autoantibodies to prothrombin are common in patients with systemic lupus erythematosus. Although their presence is a risk factor for thrombosis, neither their origin nor their precise role in inducing the procoagulant state is known. We have developed a phage-display antibody library from patients with systemic lupus erythematosus with antiprothrombin antibodies, and we have selected two single-chain Fv antibody fragments (ScFvs) by panning on a prothrombin-coated surface. In prothrombin activation assays using purified components, these antibodies promoted prothrombin activation. These ScFvs, termed AN78 and AN129, bound to immobilized prothrombin in a concentration-dependent specific manner but not to other anionic phospholipid binding proteins such as beta2-glycoprotein I or annexin V. Phosphatidylserine-bound prothrombin, but not soluble prothrombin, inhibited the binding suggesting that the epitope is available only on immobilized prothrombin. To localize the epitope, prothrombin was treated with thrombin or factor Xa and various prothrombin activation fragments were subsequently isolated and tested in ELISA with the ScFvs. Both AN78 and AN129 bound to prethrombin I (the fragment lacking the Gla domain and the first kringle domain), to fragment 1.2 (containing Gla and the two kringle domains only) and to fragment 2 but not to thrombin, thus localizing the cognate epitope to the kringle 2 domain in prothrombin. Analysis of the cDNA sequences of these antibodies show clustered mutational patterns in the complementarity determining region, suggesting that variable domains are the products of antigen-driven B cell clonal maturation.