Patrick Andre

CD40L stabilizes arterial thrombi by a β3 integrin-dependent mechanism

CD40L, a member of the tumor necrosis factor family of ligands, plays a major role in immune responses via its receptor, CD40. Recently, CD40L has been detected on the surfaces of activated platelets and shown to activate endothelium. Here we further addressed the function of platelet CD40L. We show that absence of CD40L affects the stability of arterial thrombi and delays arterial occlusion in vivo. Infusion of recombinant soluble (rs)CD40L restored normal thrombosis, whereas rsCD40L lacking the KGD integrin-recognition sequence did not. CD40-deficient mice exhibited normal thrombogenesis. rsCD40L specifically bound to purified integrin αIIbβ3 and to activated platelets in a β3-dependent manner and induced platelet spreading. In addition, rsCD40L promoted the aggregation of either human or mouse platelets under high shear rates. Thus, CD40L appears to be an αIIbβ3 ligand, a platelet agonist, and necessary for stability of arterial thrombi.

Soluble CD40 ligand induces β3 integrin tyrosine phosphorylation and triggers platelet activation by outside-in signaling

We earlier reported that the soluble form of the CD40 ligand (sCD40L), is involved in thrombosis by stabilizing platelet thrombi. In this article, we have determined the mechanism by which this protein affects platelet biology. Addition of sCD40L to washed platelets was found to activate the receptor function of αIIbβ3 as measured by the induction of fibrinogen binding and the formation of platelet microparticles. Mutation in the KGD sequence (D117E) of sCD40L, the αIIbβ3-binding domain in the N terminus of the protein resulted in a loss of the platelet-stimulatory activity of this protein. Integrilin, a αIIbβ3 antagonist, but not an antibody to CD40 that blocked the ligand-binding activity, inhibited these platelet-stimulatory events. CD40-/- platelets bound fibrinogen and formed microparticles similar to WT platelets, again indicating that CD40 is not involved in sCD40L-induced platelet activation. Exposure of platelets to sCD40L, but not D117E-sCD40L-coated surfaces, induced platelet thrombi formation under arterial shear rate. sCD40L-induced platelet stimulation resulted in the phosphorylation of tyrosine-759 in the cytoplasmic domain of β3. Platelets from the diYF mouse strain, expressing β3 in which both cytoplasmic tyrosines are mutated to phenylalanine, were defective in sCD40L-induced platelet stimulation. These data indicate that sCD40L is a primary platelet agonist and that platelet stimulation is induced by the binding of the KGD domain of sCD40L to αIIbβ3, triggering outside-in signaling by tyrosine phosphorylation of β3.

Leukocyte Engagement of Platelet Glycoprotein Ibα via the Integrin Mac-1 Is Critical for the Biological Response to Vascular Injury

Background— Leukocyte-platelet interactions are critical in the initiation and progression of atherosclerosis as well as restenosis. Although the leukocyte integrin Mac-1 (αMβ2, CD11b/CD18) has been implicated in the firm adhesion and transmigration of leukocytes at sites of platelet deposition, the precise αMβ2 counterligand responsible for mediating adhesion-strengthening interactions between neutrophils and platelets in vivo has not previously been identified. Methods and Results— Our previous studies have established the P201-K217 sequence in the αMI domain as the binding site for platelet glycoprotein (GP) Ibα. Here we report that antibody targeting of αM(P201-K217) reduced αMβ2-dependent adhesion to GP Ibα but not other αMβ2 ligands, including fibrinogen, intercellular adhesion molecule-1, and junctional adhesion molecule-3. Anti-αM(P201-K217) inhibited the firm adhesion of both human and murine leukocytes to adherent platelets under laminar flow conditions. In a mouse femoral artery wire injury model, antibody targeting of αM(P201-K217) reduced leukocyte accumulation after injury that was accompanied by inhibition of cellular proliferation and neointimal thickening. Conclusions— This study demonstrates that GP Ibα is a physiologically relevant ligand for αMβ2 and that integrin engagement of GP Ibα is critical to leukocyte function and the biological response to vascular injury. These observations establish a molecular target for selectively disrupting leukocyte-platelet complexes that promote inflammation in thrombosis and restenosis.

Defect in regulated secretion of P-selectin affects leukocyte recruitment in von Willebrand factor-deficient mice

Stimulation of endothelial cells by various inflammatory mediators leads to release of Weibel–Palade bodies and therefore to exocytosis of both P-selectin (adhesion receptor for leukocytes) and von Willebrand factor (vWf) (platelet ligand). The potential role of vWf in leukocyte recruitment was investigated with the use of vWf-deficient mice. We report a strong reduction of leukocyte rolling in venules of vWf-deficient mice. Similarly, vWf deficiency led to a decrease in neutrophil recruitment in a cytokine-induced meningitis model as well as in early skin wounds. In all instances with an antibody that preferentially recognizes plasma membrane P-selectin, we observed a dramatic reduction in P-selectin expression at the cell surface of vWf-deficient endothelium. With confocal microscopy, we found that the typical rodlike shape of the Weibel–Palade body is missing in vWf −/− endothelial cells and that part of the P-selectin content in the vWf −/− cells colocalized with LAMP-1, a lysosomal marker. However, intracellular P-selectin levels were similar in tumor necrosis factor α- and lipopolysaccharide-activated cells of both genotypes. We conclude that the absence of vWf, as found in severe von Willebrand disease, leads to a defect in Weibel–Palade body formation. This defect results in decreased P-selectin translocation to the cell surface and reduced leukocyte recruitment in early phases of inflammation.

P‐selectin in haemostasis

During the past decade, interrelationships between inflammation and thrombosis have been the subject of extensive works, and it is now commonly recognized that inflammation (notably leucocyte recruitment) directly affects thrombosis, and that thrombosis also constitutes a pro‐inflammatory event. This tight link is partly attributable to P‐selectin, which is functional not only when expressed on the surface of activated platelets and endothelial cells, but also when shed, generating its soluble form, termed sP‐selectin. In this review, we will provide an overview of the relative roles of the different compartments of P‐selectin (platelet, endothelial cell, plasma) in haemostasis and vascular pathologies, and the potential therapeutic benefits achievable in targeting this molecule.

ApoE Deficiency Compromises the Blood Brain Barrier Especially After Injury

BackgroundApolipoprotein E (apoE) mediates lipoprotein uptake by receptors such as the LDL receptor (LDLR). The isoform apoE4 has been linked to Alzheimer’s disease and to poor outcomes after brain injury. Astrocytes that induce blood brain barrier (BBB) properties in endothelium also produce apoE. We decided to investigate the role of apoE in BBB function and in the restoration of BBB after brain injury.Materials and MethodsWild-type (WT) mice and mice deficient in apoE or LDLR were fed normal chow or diets rich in fat and cholesterol. The BBB leakage was determined through injection of Evans blue dye and measurement of the amount of dye extravasated in the brains 3 hours later. Brain injury was induced by applying dry ice directly onto the excised parietal region of the brain. The mice were given 7 days to recover. In some experiments, peroxidase was infused to observe the site of leakage by histology.ResultsWe found 70% more spontaneous leakage of injected Evans blue dye in the brains of apoE−/− mice than in wild type. This increase in permeability appeared selective for the brain. The leaky BBB in apoE−/− mice may provide an explanation for the neurological deficits seen in these animals. In an established model of BBB leakage induced by trauma (cold injury), the apoE−/− mice showed even more compromised BBB function, compared with WT mice, suggesting that apoE is important for BBB recovery. No deficit in BBB was observed in injured LDLR−/− mice, even on Western Diet. In contrast, higher plasma cholesterol levels in apoE−/− mice further increased BBB leakage after injury. We extracted 5x more Evans blue from these brains than from WT. In the injury model, injection of peroxidase resulted in prominent retention of this protein in the cortex of apoE−/− but not in WT.ConclusionsOur results show that the combination of loss of apoE function with high plasma cholesterol and especially brain injury results in dramatic BBB defects in the cortex and may explain in part the importance of apoE in Alzheimer’s disease and in successful recovery from brain injury.

The platelet CD40L/GP IIb-IIIa axis in atherothrombotic disease.

Platelet aggregation, a process mediated by GP IIb-IIIa, is responsible for the occlusive events in thrombosis: indeed, GP IIb-IIIa antagonists are effective in blocking arterial thrombosis. Recent studies have suggested that activated platelets and platelet thrombi can contribute significantly to the initiation and progression of atherosclerosis, a chronic inflammatory disease. Platelets store inflammatory cytokines such as CD40L and RANTES, which are now known to be important in this pathologic process. CD40L appears to be particularly relevant because high levels of the circulating soluble form of CD40L, termed sCD40L, are released in response to platelet thrombosis and because elevated levels of sCD40L are a reliable predictor of cardiovascular events. sCD40L is also a ligand of GP IIb-IIIa and is involved in thrombus stabilization and platelet activation. In addition, GP IIb-IIIa antagonists unexpectedly block release of sCD40L. These observations suggest that long-term benefit of GP IIb-IIIa antagonists treatment could be due not only to the inhibition of the acute ischemic complications, but also to the inhibition of autocrine function of sCD40L, thereby interrupting the platelet CD40L/GP IIb-IIIa axis.

Rap1-Rac1 Circuits Potentiate Platelet Activation

Objective—The goal of this study was to investigate the potential crosstalk between Rap1 and Rac1, 2 small GTPases central to platelet activation, particularly downstream of the collagen receptor GPVI. Methods and Results—We compared the activation response of platelets with impaired Rap signaling (double knock-out; deficient in both the guanine nucleotide exchange factor, CalDAG-GEFI, and the Gi-coupled receptor for ADP, P2Y12), to that of wild-type platelets treated with a small-molecule Rac inhibitor, EHT 1864 (wild-type /EHT). We found that Rac1 is sequentially activated downstream of Rap1 on stimulation via GPVI. In return, Rac1 provides important feedback for both CalDAG-GEFI– and P2Y12-dependent activation of Rap1. When analyzing platelet responses controlled by Rac1, we observed (1) impaired lamellipodia formation, clot retraction, and granule release in both double knock-out and EHT 1864-treated wild-type platelets; and (2) reduced calcium store release in EHT 1864-treated wild-type but not double knock-out platelets. Consistent with the latter finding, we identified 2 pools of Rac1, one activated immediately downstream of GPVI and 1 activated downstream of Rap1. Conclusion—We demonstrate important crosstalk between Rap1 and Rac1 downstream of GPVI. Whereas Rap1 signaling directly controls sustained Rac1 activation, Rac1 affects CalDAG-GEFI– and P2Y12-dependent Rap1 activation via its role in calcium mobilization and granule/ADP release, respectively.

Platelet-derived S100 family member myeloid-related protein-14 regulates thrombosis.

Expression of the gene encoding the S100 calcium-modulated protein family member MRP-14 (also known as S100A9) is elevated in platelets from patients presenting with acute myocardial infarction (MI) compared with those from patients with stable coronary artery disease; however, a causal role for MRP-14 in acute coronary syndromes has not been established. Here, using multiple models of vascular injury, we found that time to arterial thrombotic occlusion was markedly prolonged in Mrp14⁻/⁻ mice. We observed that MRP-14 and MRP-8/MRP-14 heterodimers (S100A8/A9) are expressed in and secreted by platelets from WT mice and that thrombus formation was reduced in whole blood from Mrp14⁻/⁻ mice. Infusion of WT platelets, purified MRP-14, or purified MRP-8/MRP-14 heterodimers into Mrp14⁻/⁻ mice decreased the time to carotid artery occlusion after injury, indicating that platelet-derived MRP-14 directly regulates thrombosis. In contrast, infusion of purified MRP-14 into mice deficient for both MRP-14 and CD36 failed to reduce carotid occlusion times, indicating that CD36 is required for MRP-14-dependent thrombosis. Our data identify a molecular pathway of thrombosis that involves platelet MRP-14 and CD36 and suggest that targeting MRP-14 has potential for treating atherothrombotic disorders, including MI and stroke.

Critical role for Syk in responses to vascular injury

Although current antiplatelet therapies provide potent antithrombotic effects, their efficacy is limited by a heightened risk of bleeding and failure to affect vascular remodeling after injury. New lines of research suggest that thrombosis and hemorrhage may be uncoupled at the interface of pathways controlling thrombosis and inflammation. Here, as one remarkable example, studies using a novel and highly selective pharmacologic inhibitor of the spleen tyrosine kinase Syk [PRT060318; 2-((1R,2S)-2-aminocyclohexylamino)-4-(m-tolylamino)pyrimidine-5-carboxamide] coupled with genetic experiments, demonstrate that Syk inhibition ameliorates both the acute and chronic responses to vascular injury without affecting hemostasis. Specifically, lack of Syk (murine radiation chimeras) attenuated shear-induced thrombus formation ex vivo, and PRT060318 strongly inhibited arterial thrombosis in vivo in multiple animal species while having minimal impact on bleeding. Furthermore, leukocyte-platelet-dependent responses to vascular injury, including inflammatory cell recruitment and neointima formation, were markedly inhibited by PRT060318. Thus, Syk controls acute and long-term responses to arterial vascular injury. The therapeutic potential of Syk may be exemplary of a new class of antiatherothrombotic agents that target the interface between thrombosis and inflammation.