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 (

Key Points• Temporal profiles of .4000 phosphopeptides after stimulating human platelets (a) with ADP and (b) consecutively with ADP and Iloprost.• Reciprocal phosphorylation profiles of ADP and Iloprost point to central players of platelet homeostasis.Adenosine diphosphate (ADP) enhances platelet activation by virtually any other stimulant to complete aggregation. It binds specifically to the G-protein-coupled membrane receptors P2Y1 and P2Y12, stimulating intracellular signaling cascades, leading to integrin aIIbb3 activation, a process antagonized by endothelial prostacyclin. P2Y12 inhibitors are among the most successful antiplatelet drugs, however, show remarkable variability in efficacy. We reasoned whether a more detailed molecular understanding of ADP-induced protein phosphorylation could identify (1) critical hubs in platelet signaling toward aggregation and (2) novel molecular targets for antiplatelet treatment strategies. We applied quantitative temporal phosphoproteomics to study ADP-mediated signaling at unprecedented molecular resolution. Furthermore, to mimic the antagonistic efficacy of endothelial-derived prostacyclin, we determined how Iloprost reverses ADP-mediated signaling events. We provide temporal profiles of 4797 phosphopeptides, 608 of which showed significant regulation. Regulated proteins are implicated in well-known activating functions such as degranulation and cytoskeletal reorganization, but also in less well-understood pathways, involving ubiquitin ligases and GTPase exchange factors/GTPaseactivating proteins (GEF/GAP). Our data demonstrate that ADP-triggered phosphorylation occurs predominantly within the first 10 seconds, with many short rather than sustained changes. For a set of phosphorylation sites (eg, PDE3A Ser312 , CALDAG-GEFI Ser587 , ENSA Ser109 ), we demonstrate an inverse regulation by ADP and Iloprost, suggesting that these are central modulators of platelet homeostasis. This study demonstrates an extensive spectrum of human platelet protein phosphorylation in response to ADP and Iloprost, which inversely overlap and represent major activating and inhibitory pathways. (Blood. 2017;129(2):e1-e12)

Protein kinase A (PKA) activation by cAMP phosphorylates multiple target proteins in numerous platelet inhibitory pathways that have a very important role in maintaining circulating platelets in a resting state. Here we show that in thrombin-and collagen-stimulated platelets, PKA is activated by cAMP-independent mechanisms involving dissociation of the catalytic subunit of PKA (PKAc) from an NFB-IB␣-PKAc complex. We demonstrate mRNA and protein expression for most of the NFB family members in platelets. From resting platelets, PKAc was co-immunoprecipitated with IB␣, and conversely, IB␣ was also co-immunoprecipitated with PKAc. This interaction was significantly reduced in thrombin-and collagen-stimulated platelets. Stimulation of platelets with thrombin-or collagen-activated IKK, at least partly by PI3 kinase-dependent pathways, leading to phosphorylation of IB␣, disruption of an IB␣-PKAc complex, and release of free, active PKAc, which phosphorylated VASP and other PKA substrates. IKK inhibitor inhibited thrombin-stimulated IkB␣ phosphorylation, PKAIkB␣ dissociation, and VASP phosphorylation, and potentiated integrin ␣IIb␤3 activation and the early phase of platelet aggregation. We conclude that thrombin and collagen not only cause platelet activation but also appear to fine-tune this response by initiating downstream NFB-dependent PKAc activation, as a novel feedback inhibitory signaling mechanism for preventing undesired platelet activation.Platelets are small anucleate cells derived from megakaryocytes in the bone marrow, in a process in which megakaryocyte cytoplasmic extensions into microvessels are sheared from their transendothelial stems by flowing blood (1-2). Platelets play a key role in the normal homeostatic process through their ability to rapidly adhere to activated and/or injured endothelium and subendothelial matrix proteins (platelet adhesion), and to other activated platelets (platelet aggregation). Many factors bind to specific platelet receptors and regulate signaling pathways, which promote or inhibit platelet adhesion, aggregation, and secretion. In vivo, circulating platelets are continually exposed to a variety of activating factors including collagen, fibrinogen, ADP, von Willebrand Factor (vWF), thrombin, and thromboxane (3-5), as well as inhibitory factors such as endothelial-derived nitric oxide (NO), prostacyclin (PG-I 2 ), and ADPase (3, 5-6). A strong equilibrium between the two opposing processes of platelet stimulation and inhibition is thought to be essential for normal platelet and vascular function. An impairment of this equilibrium will promote either thrombotic or bleeding disorders.In the initial steps of platelet activation, the platelet receptor glycoproteins (GP) 3 1b and GPVI interact with extracellular matrix (ECM) proteins, causing platelets to tether and roll on the injured endothelium or subendothelial ECM (5). Stimulation of these receptors triggers intracellular signaling cascades that activate integrin ␣IIb␤3 and induce the release of secondary mediators like A...

Abstract-Clopidogrel is an effective new antiplatelet agent useful for the treatment of ischemic cerebrovascular, cardiac, and peripheral arterial disease. However, the mechanism of clopidogrel action is not well understood, although it is known to inhibit ADP-evoked platelet aggregation. In the current study, the effect of clopidogrel on recently identified human platelet ADP receptors and their signaling pathways was investigated by using platelets from clopidogrel-treated subjects, 6 healthy volunteers (2 females and 4 males) who received 75 mg of clopidogrel daily for 7 days. Blood was taken and various platelet receptor signaling pathways were analyzed before treatment, after 7 days of medication, and 4 weeks after treatment had ceased. Platelet tests included the analysis of aggregation, rapid calcium influx, calcium mobilization from intracellular stores, adenylyl cyclase, and phosphorylation of vasodilator-stimulated phosphoprotein (VASP). The data indicate that clopidogrel does not affect those platelet ADP receptors coupled to cation influx (P2X1 ADP receptors) or calcium mobilization (P2Y1 ADP receptors). In contrast, clopidogrel treatment specifically impairs the ADP receptor coupled to G i /adenylyl cyclase (P2Y AC ADP receptors). Clopidogrel abolishes the inhibitory P2Y AC receptor-mediated ADP effects on prostaglandin E 1 -stimulated, cAMP-dependent phosphorylation of VASP without affecting epinephrine, thrombin, and thromboxane signaling. VASP phosphorylation is known to be closely correlated with the inhibition of platelet and fibrinogen receptor (glycoprotein IIb/IIIa) activation. Therefore, inhibition of the platelet P2Y AC ADP receptor and its intracellular signaling, including decreased VASP phosphorylation, is suggested as a molecular mechanism of clopidogrel action. Key Words: platelet inhibition Ⅲ purinergic receptors Ⅲ vasodilator-stimulated phosphoprotein I ncreased platelet activation and aggregation are central to the pathophysiology of acute and chronic arterial vascular diseases. This concept has gained broad acceptance since platelet inhibitors have been proven as effective agents for the treatment of both chronic and acute diseases of the arterial vessel wall. [1][2][3][4][5][6] Platelets are activated by numerous agents and conditions, but ADP is thought to play a key role in the development of arterial thrombosis. 7,8 Recently, long-term administration of clopidogrel to patients with atherosclerotic vascular disease was shown to be more effective than aspirin in reducing the combined risk of ischemic stroke, myocardial infarction, and vascular death. 9 Clopidogrel and the chemically related ticlopidine are thienopyridines that selectively and specifically interfere with ADP-mediated platelet activation. 5,10 In contrast to ticlopidine, which may cause neutropenia, clopidogrel appears to be a safe and well-tolerated drug. 9 Thienopyridines are inactive in-vitro, require in vivo metabolism, and cause an irreversible inhibition of platelet function. However, the mechanism of thieno...