Loss of oligophrenin1 leads to uncontrolled Rho activation and increased thrombus formation in mice.

BACKGROUND: Platelet cytoskeletal reorganization is essential for platelet adhesion and thrombus formation in hemostasis and thrombosis. The Rho GTPases RhoA, Rac1 and Cdc42 are the main players in cytoskeletal dynamics of platelets and induce filopodia and lamellipodia formation and actin polymerization to strongly increase the platelet surface upon activation. Moreover, they are important for platelet secretion, integrin activation and arterial thrombus formation. OBJECTIVES: Rho GTPases are regulated by GTPase-activating proteins (GAPs) that stimulate their GTPase activity to terminate Rho signaling. The regulation of Rho GTPase activity in platelets is not well defined. Recently, we identified oligophrenin1 (OPHN1), a RhoGAP in platelets that exhibits strong GTPase-stimulating activity towards RhoA, Cdc42 and Rac1. RESULTS: In the present study we show for the first time, that deficiency of OPHN1 led to abnormal Rho activation and increased platelet cytoskeletal reorganization, including cell adhesion and lamellipodia formation on fibrinogen. Furthermore, platelets from ophn1(-/-) mice showed enhanced susceptibility to platelet activation with alterations in actin distribution and early release of granules. Platelet activation was enhanced following GPVI and PAR4 stimulation. This translated into elevated platelet thrombus formation and promoted arterial thrombosis under low shear conditions with altered hemostasis, as detected by tail bleeding time. CONCLUSIONS: The results of the present study identified OPHN1 as an important regulator of platelet cytoskeletal reorganization and demonstrate that abnormal regulation of Rho proteins leads to increased platelet adhesion and thrombus formation under low shear conditions in vitro and in vivo, suggesting a prothrombotic phenotype of mice critical for acute thrombotic occlusions.

Nadrin GAP activity is isoform- and target-specific regulated by tyrosine phosphorylation.

Cytoskeletal reorganization is crucial for platelet adhesion and thrombus formation to avoid excessive bleeding. Major regulators of cytoskeletal dynamics are small GTPases of the Rho family. Rho GTPases become activated by G-protein coupled receptor activation, downstream of immunoreceptor tyrosine-based activation motif (ITAM)-coupled receptors and by outside-in signaling of integrins. They act as molecular switches and cycle between active and inactive states. GTPase activating proteins (GAPs) stimulate the hydrolysis of GTP to GDP to terminate Rho signaling. Nadrin is a RhoGAP that was recently identified in platelets. Five Nadrin isoforms are known consisting of a unique GAP and an N-terminal BAR domain responsible for the selective regulation of RhoA, Cdc42 and Rac1. Besides BAR domain mediated regulation of Nadrin GAP activity nothing is known about the regulation of Nadrin and the impact on cytoskeletal reorganization. Here we show that Nadrin becomes tyrosine phosphorylated upon platelet activation. We found Src family proteins (Src, Lyn, Fyn) to be responsible to control Nadrin GAP activity by phosphorylation. Interestingly, phosphorylation of Nadrin leads to tightly regulated Rho activation that was found to be Nadrin isoform- and (Rho) target-specific. Src-phosphorylation of Nadrin5 mediated inactivation of Cdc42 while RhoA and Rac1 became activated upon Src-mediated phosphorylation of Nadrin2. Our results suggest a critical role for spatial and temporal regulation of Nadrin and thus for the control of Rho GTPases in platelets.

Isoform-specific roles of the GTPase activating protein Nadrin in cytoskeletal reorganization of platelets.

Cytoskeletal reorganization of activated platelets plays a crucial role in hemostasis and thrombosis and implies activation of Rho GTPases. Rho GTPases are important regulators of cytoskeletal dynamics and function as molecular switches that cycle between an inactive and an active state. They are regulated by GTPase activating proteins (GAPs) that stimulate GTP hydrolysis to terminate Rho signaling. The regulation of Rho GTPases in platelets is not explored. A detailed characterization of Rho regulation is necessary to understand activation and inactivation of Rho GTPases critical for platelet activation and aggregation. Nadrin is a RhoGAP regulating cytoplasmic protein explored in the central nervous system. Five Nadrin isoforms are known that share a unique GAP domain, a serine/threonine/proline-rich domain, a SH3-binding motif and an N-terminal BAR domain but differ in their C-terminus. Here we identified Nadrin in platelets where it co-localizes to actin-rich regions and Rho GTPases. Different Nadrin isoforms selectively regulate Rho GTPases (RhoA, Cdc42 and Rac1) and cytoskeletal reorganization suggesting that - beside the GAP domain - the C-terminus of Nadrin determines Rho specificity and influences cell physiology. Furthermore, Nadrin controls RhoA-mediated stress fibre and focal adhesion formation. Spreading experiments on fibrinogen revealed strongly reduced cell adhesion upon Nadrin overexpression. Unexpectedly, the Nadrin BAR domain controls Nadrin-GAP activity and acts as a guidance domain to direct this GAP to its substrate at the plasma membrane. Our results suggest a critical role for Nadrin in the regulation of RhoA, Cdc42 and Rac1 in platelets and thus for platelet adhesion and aggregation.

Causes of infertility in men with Down syndrome.

Men with Down syndrome are considered as infertile although the causes of infertility are not known in detail yet. Although this constitutes a general rule there are three confirmed cases of parenting by fathers with Down syndrome. Many investigators have addressed the causes of infertility and their studies indicate that the causes may be hormonal deficits, morphological alterations of the gonads, abnormal spermatogenesis, psychological and social factors related to the mental retardation. It is obvious that the extra chromosome 21 has a detrimental direct and indirect effect on the reproductive capacity of the affected male patient. But the definite cause of the insufficient and inadequate spermatogenesis remains to be discovered.

Assessment of intraocular foreign bodies by helical-CT multiplanar imaging.

The aim of this study was to examine the effectiveness of helical CT in the assessment of intraocular foreign bodies, evaluating two protocols with different collimation. We performed helical-CT studies in 30 patients. Fifteen patients were examined with 1.5-mm collimation and the other 15 patients with 3.0-mm collimation. All other imaging parameters were identical in both protocols. Multiplanar images were reconstructed. The examinations were reviewed for presence, localization and size of intraocular foreign bodies. We compare our results with the surgical data. We estimate the required examination time. In the first group (collimation 1.5 mm) an intraorbital foreign body was detected in 8 of 15 patients. In 3 of 8 patients an intraocular foreign body (all were metallic) was detected. In the second group (collimation 3.0 mm) an intraorbital foreign body was detected in 9 of 15 patients. In 8 of 9 patients an intraocular foreign body (all were metallic) was detected. Our results were confirmed by surgery in all cases. Examination time was 36 s in the first group and 18 s in the second group. Computed tomography should be considered the imaging modality of choice in the assessment of metallic intraocular foreign bodies and 3.0-mm collimation is optional, because of reduced examination time and radiation exposure.