Invasive cell migration through tissue barriers requires pericellular remodelling of extracellular matrix (ECM) executed by cell-surface proteases, particularly membrane-type-1 matrix metalloproteinase (MT1-MMP/MMP-14). Using time-resolved multimodal microscopy, we show how invasive HT-1080 fibrosarcoma and MDA-MB-231 breast cancer cells coordinate mechanotransduction and fibrillar collagen remodelling by segregating the anterior force-generating leading edge containing beta1 integrin, MT1-MMP and F-actin from a posterior proteolytic zone executing fibre breakdown. During forward movement, sterically impeding fibres are selectively realigned into microtracks of single-cell calibre. Microtracks become expanded by multiple following cells by means of the large-scale degradation of lateral ECM interfaces, ultimately prompting transition towards collective invasion similar to that in vivo. Both ECM track widening and transition to multicellular invasion are dependent on MT1-MMP-mediated collagenolysis, shown by broad-spectrum protease inhibition and RNA interference. Thus, invasive migration and proteolytic ECM remodelling are interdependent processes that control tissue micropatterning and macropatterning and, consequently, individual and collective cell migration.

Antiplatelet treatment is of fundamental importance in combatting functions/dysfunction of platelets in the pathogenesis of cardiovascular and inflammatory diseases. Dysfunction of anucleate platelets is likely to be completely attributable to alterations in posttranslational modifications and protein expression. We therefore examined the proteome of platelets highly purified from fresh blood donations, using elaborate protocols to ensure negligible contamination by leukocytes, erythrocytes, and plasma. Using quantitative mass spectrometry, we created the first comprehensive and quantitative human platelet proteome, comprising almost 4000 unique proteins, estimated copy numbers for similar to 3700 of those, and assessed intersubject (4 donors) as well as intrasubject (3 different blood samples from 1 donor) variations of the proteome. For the first time, our data allow for a systematic and weighted appraisal of protein networks and pathways in human platelets, and indicate the feasibility of differential and comprehensive proteome analyses from small blood donations. Because 85% of the platelet proteome shows no variation between healthy donors, this study represents the starting point for disease-oriented platelet proteomics. In the near future, comprehensive and quantitative comparisons between normal and well-defined dysfunctional platelets, or between platelets obtained from donors at various stages of chronic cardiovascular and inflammatory diseases will be feasible

SummaryIncreased platelet adhesion or aggregation are key events in the pathogenesis of cardiovascular diseases. Exact determination of the platelet activation state is essential to recognize, prevent, and treat cardiovascular complications due to platelet dysfunction. Initial phases of platelet activation and inhibition are characterized by phosphorylation of specific intracellular proteins. However, methodological problems often prevent analysis of platelet protein phosphorylation under clinical conditions. A novel flow cytometry-based method using a phosphorylation-specific antibody was developed for fast and easy quantification of the phosphorylation state of a specific intracellular platelet protein. This method was used to analyze various platelet receptors and their intracellular signaling which may be impaired in genetic or acquired disorders or altered due to therapeutic interventions. In a first clinical application, the inhibitory effects of ticlopidine and clopidogrel on the platelet P2YAC ADP receptor were monitored.Abbreviations: ADP: adenosine 5’-diphosphate; cAMP: cyclic adenosine-3’,5’-monophosphate; cGMP: cyclic guanosine-3’,5’-monophosphate; HUVECs: human umbilical vein endothelial cells; MAPK: mitogen-activated protein kinase; PG-E1: prostaglandin E1; PRP: platelet-rich plasma; SNP: sodium nitroprusside; VASP: vasodilator-stimulated phosphoprotein

A BSTR ACTVasodilator-stimulated phosphoprotein (VASP), a substrate of cAMP-and cGMP-dependent protein kinases, is associated with focal adhesions, cell-cell contacts, microfilaments, and highly dynamic membrane regions. VASP, which is expressed in most cell types and in particularly high levels in human platelets, binds to profilin, zyxin, vinculin, F-actin, and the Listeria monocytogenes surface protein ActA. VASP is a member of the enabled (Ena)͞VASP protein family and is thought to be involved in actin filament formation and integrin ␣ IIb ␤ 3 inhibition in human platelets. To gain further insight into the in vivo function of this protein, VASP-deficient mice were generated by homologous recombination. VASP؊͞؊ mice demonstrated hyperplasia of megakaryocytes in bone marrow and spleen but exhibited no other macroscopic or microscopic abnormalities. Activation of platelets with thrombin induced a more than 2-fold higher surface expression of P-selectin and fibrinogen binding in VASP-deficient platelets in comparison to wild type. These data support the concept that VASP is a negative modulator of platelet and integrin ␣ IIb ␤ 3 activation. Although the limited phenotypic differences between wild-type and VASP؊͞؊ mice suggested functional compensation of VASP by members of the Ena͞VASP family, alterations in the expression levels of mammalian enabled (Mena) and Ena-VASP-like (Evl) protein were not detected. VASP-deficient mice may provide an interesting model system for diseases in which enhanced platelet activation plays a major role.Vasodilator-stimulated phosphoprotein (VASP) initially was discovered and characterized as a prominent substrate for both cGMP-dependent protein kinases (cGKs) and cAMP-dependent protein kinases (cAKs) in human platelets (1, 2). VASP is phosphorylated in vitro and in intact human platelets at serine-157, serine-239, and threonine-278 by both cAK and cGK, and it is in vitro and in intact human platelet dephosphorylated by protein phosphatases I and II with overlapping selectivity (3-7). Phosphorylation of serine-157, the site preferred by cAK, leads to a shift in the apparent molecular mass of VASP in SDS͞PAGE from 46 kDa to 50 kDa (5, 6). VASP phosphorylation in response to cyclic nucleotide-regulating vasodilators (i.e., cAMP-elevating prostaglandins and cGMP-elevating NO donors) closely correlates with platelet inhibition and in particular with the inhibition of fibrinogen binding to the human platelet integrin ␣ IIb ␤ 3 (3,8).

Platelets play a crucial role in the physiology of primary hemostasis and pathophysiologic processes such as arterial thrombosis. Accumulating evidence suggests a role of reactive oxygen species (ROSs) in platelet activation. Here we show that platelets activated with different agonists produced intracellular ROSs, which were reduced by reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) oxidase inhibitors and superoxide scavengers. In addition, we demonstrate that ROSs produced in platelets significantly affected ␣IIb␤3 integrin activation but not alpha and dense granule secretion and platelet shape change. Thrombin-induced integrin ␣IIb␤3 activation was significantly decreased after pretreatment of platelets with NAD (