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Background: Platelets play an important role in cardiovascular and cerebrovascular diseases. Abdominal aortic aneurysm (AAA) is a highly lethal, atherosclerosis-related disease with characteristic features of progressive dilatation of the abdominal aorta and degradation of the vessel wall, accompanied by chronic inflammation. Platelet activation and procoagulant activity play a decisive role in the AAA pathology as they might trigger AAA development in both mice and humans., Methods: The present study investigated the impact of the major platelet collagen receptor GP (platelet glycoprotein) VI in pathophysiological processes underlying AAA initiation and progression. For experimental AAA induction in mice, PPE (porcine pancreatic elastase) and the external PPE model were used., Results: Genetic deletion of GP VI offered protection of mice against aortic diameter expansion in experimental AAA. Mechanistically, GP VI deficiency resulted in decreased inflammation with reduced infiltration of neutrophils and platelets into the aortic wall. Furthermore, remodeling of the aortic wall was improved in the absence of GP VI, as indicated by reduced MMP (matrix metalloproteinase)-2/9 and OPN (osteopontin) plasma levels and an enhanced α-SMA (α-smooth muscle actin) content within the aortic wall, accompanied by reduced cell apoptosis. Consequently, an elevation in intima/media thickness and elastin content was observed in GP VI-deficient PPE mice, resulting in a significantly reduced aortic diameter expansion and reduced aneurysm incidence. In patients with AAA, enhanced plasma levels of soluble GP VI and fibrin, as well as fibrin accumulation within the intraluminal thrombus might serve as new biomarkers to detect AAA early. Moreover, we hypothesize that GP VI might play a role in procoagulant activity and thrombus stabilization via binding to fibrin., Conclusions: In conclusion, our results emphasize the potential need for a GP VI-targeted antiplatelet therapy to reduce AAA initiation and progression, as well as to protect patients with AAA from aortic rupture., Competing Interests: None.

Objective: Reelin, a secreted glycoprotein, was originally identified in the central nervous system, where it plays an important role in brain development and maintenance. In the cardiovascular system, reelin plays a role in atherosclerosis by enhancing vascular inflammation and in arterial thrombosis by promoting platelet adhesion, activation, and thrombus formation via APP (amyloid precursor protein) and GP (glycoprotein) Ib. However, the role of reelin in hemostasis and arterial thrombosis is not fully understood to date. Approach and Results: In the present study, we analyzed the importance of reelin for cytoskeletal reorganization of platelets and thrombus formation in more detail. Platelets release reelin to amplify alphaIIb beta3 integrin outside-in signaling by promoting platelet adhesion, cytoskeletal reorganization, and clot retraction via activation of Rho GTPases RAC1 (Ras-related C3 botulinum toxin substrate) and RhoA (Ras homolog family member A). Reelin interacts with the collagen receptor GP (glycoprotein) VI with subnanomolar affinity, induces tyrosine phosphorylation in a GPVI-dependent manner, and supports platelet binding to collagen and GPVI-dependent RAC1 activation, PLC gamma 2 (1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase gamma-2) phosphorylation, platelet activation, and aggregation. When GPVI was deleted from the platelet surface by antibody treatment in reelin-deficient mice, thrombus formation was completely abolished after injury of the carotid artery while being only reduced in either GPVI-depleted or reelin-deficient mice., Conclusions: Our study identified a novel signaling pathway that involves reelin-induced GPVI activation and alphaIIb beta3 integrin outside-in signaling in platelets. Loss of both, GPVI and reelin, completely prevents stable arterial thrombus formation in vivo suggesting that inhibiting reelin-platelet-interaction might represent a novel strategy to avoid arterial thrombosis in cardiovascular disease.

Objective: Thrombin signaling promotes atherosclerosis by initiating inflammatory events indirectly through platelet activation and directly via protease-activated receptors. Therefore, endogenous thrombin inhibitors may be relevant modulators of atheroprogression and cardiovascular risk. In addition, endogenous thrombin inhibitors may affect the response to non-vitamin K-dependent oral anticoagulants. Here, the question was addressed whether the small leucine-rich proteoglycan biglycan acts as an endogenous thrombin inhibitor in atherosclerosis through activation of heparin cofactor II., Approach and Results: Biglycan concentrations were elevated in the plasma of patients with acute coronary syndrome and in male Apolipoprotein E-deficient (ApoE(-/-)) mice. Biglycan was detected in the glycocalyx of capillaries and the subendothelial matrix of arterioles of ApoE(-/-) mice and in atherosclerotic plaques. Thereby a vascular compartment is provided that may mediate the endothelial and subendothelial activation of heparin cofactor II through biglycan. ApoE and Bgn double-deficient (ApoE(-/-)/Bgn(-/0)) mice showed higher activity of circulating thrombin, increased platelet activation and platelet adhesion in vivo, supporting a role of biglycan in balancing thrombin activity. Furthermore, concentrations of circulating cytokines and aortic macrophage content were elevated in ApoE(-/-)/Bgn(-/0) mice, suggesting a proinflammatory phenotype. Elevated platelet activation and macrophage accumulation were reversed by treating ApoE(-/-)/Bgn(-/0) mice with the thrombin inhibitor argatroban. Ultimately, ApoE(-/-)/Bgn(-/0) mice developed aggravated atherosclerosis., Conclusions: The present results indicate that biglycan plays a previously unappreciated protective role during the progression of atherosclerosis by inhibiting thrombin activity, platelet activation, and finally macrophage-mediated plaque inflammation., (© 2016 American Heart Association, Inc.)

Objective: Platelet activation is essential for primary hemostasis and acute thrombotic vascular occlusions. On activation, platelets release their prothrombotic granules and expose phosphatidylserine, thus fostering thrombin generation and thrombus formation. In other cell types, both degranulation and phosphatidylserine exposure are modified by sphingomyelinase-dependent formation of ceramide. The present study thus explored whether acid sphingomyelinase participates in the regulation of platelet secretion, phosphatidylserine exposure, and thrombus formation., Approach and Results: Collagen-related peptide-induced or thrombin-induced ATP release and P-selectin exposure were significantly blunted in platelets from Asm-deficient mice (Smpd1(-/-)) when compared with platelets from wild-type mice (Smpd1(+/+)). Moreover, phosphatidylserine exposure and thrombin generation were significantly less pronounced in Smpd1(-/-) platelets than in Smpd1(+/+) platelets. In contrast, platelet integrin αIIbβ3 activation and aggregation, as well as activation-dependent Ca(2+) flux, were not significantly different between Smpd1(-/-) and Smpd1(+/+) platelets. In vitro thrombus formation at shear rates of 1700 s(-1) and in vivo thrombus formation after FeCl3 injury were significantly blunted in Smpd1(-/-) mice while bleeding time was unaffected. Asm-deficient platelets showed significantly reduced activation-dependent ceramide formation, whereas exogenous ceramide rescued diminished platelet secretion and thrombus formation caused by Asm deficiency. Treatment of Smpd1(+/+) platelets with bacterial sphingomyelinase (0.01 U/mL) increased, whereas treatment with functional acid sphingomyelinase-inhibitors, amitriptyline or fluoxetine (5 μmol/L), blunted activation-dependent platelet degranulation, phosphatidylserine exposure, and thrombus formation. Impaired degranulation and thrombus formation of Smpd1(-/-) platelets were again overcome by exogenous bacterial sphingomyelinase., Conclusions: Acid sphingomyelinase is a completely novel element in the regulation of platelet plasma membrane properties, secretion, and thrombus formation.

Background: CXCR4-positive bone marrow cells (BMCs) are critically involved in cardiac repair mechanisms contributing to preserved cardiac function. Stromal cell-derived factor-1 (SDF-1) is the most prominent BMC homing factor known to augment BMC engraftment, which is a limiting step of stem cell-based therapy. After myocardial infarction, SDF-1 expression is rapidly upregulated and promotes myocardial repair., Methods and Results: We have established a bifunctional protein consisting of an SDF-1 domain and a glycoprotein VI (GPVI) domain with high binding affinity to the SDF-1 receptor CXCR4 and extracellular matrix proteins that become exposed after tissue injury. SDF1-GPVI triggers chemotaxis of CXCR4-positive cells, preserves cell survival, enhances endothelial differentiation of BMCs in vitro, and reveals proangiogenic effects in ovo. In a mouse model of myocardial infarction, administration of the bifunctional protein leads to enhanced recruitment of BMCs, increases capillary density, reduces infarct size, and preserves cardiac function., Conclusions: These results indicate that administration of SDF1-GPVI may be a promising strategy to treat myocardial infarction to promote myocardial repair and to preserve cardiac function.