Spatial distribution of factor Xa, thrombin, and fibrin(ogen) on thrombi at venous shear.

Background: The generation of thrombin is a critical process in the formation of venous thrombi. In isolated plasma under static conditions, phosphatidylserine (PS)-exposing platelets support coagulation factor activation and thrombin generation; however, their role in supporting coagulation factor binding under shear conditions remains unclear. We sought to determine where activated factor X (FXa), (pro)thrombin, and fibrin(ogen) are localized in thrombi formed under venous shear.

Platelet response heterogeneity in thrombus formation.

Vascular injury leads to formation of a structured thrombus as a consequence of platelet activation and aggregation, thrombin and fibrin formation, and trapping of leukocytes and red cells. This review summarises current evidence for heterogeneity of platelet responses and functions in the thrombus-forming process. Environmental factors contribute to response heterogeneity, as the platelets in a thrombus adhere to different substrates, and sense specific (ant)agonists and rheological conditions.

Dual role of collagen in factor XII-dependent thrombus formation.

In vivo mouse models have indicated that the intrinsic coagulation pathway, initiated by factor XII, contributes to thrombus formation in response to major vascular damage. Here, we show that fibrillar type I collagen provoked a dose-dependent shortening of the clotting time of human plasma via activation of factor XII. This activation was mediated by factor XII binding to collagen.

The tyrosine phosphatase CD148 is an essential positive regulator of platelet activation and thrombosis.

Platelets play a fundamental role in hemostasis and thrombosis. They are also involved in pathologic conditions resulting from blocked blood vessels, including myocardial infarction and ischemic stroke. Platelet adhesion, activation, and aggregation at sites of vascular injury are regulated by a diverse repertoire of tyrosine kinase-linked and G protein-coupled receptors.

Differential roles of the PKC novel isoforms, PKCdelta and PKCepsilon, in mouse and human platelets.

Background: Increasing evidence suggests that individual isoforms of protein kinase C (PKC) play distinct roles in regulating platelet activation.

Methodology/principal Findings: In this study, we focus on the role of two novel PKC isoforms, PKCdelta and PKCepsilon, in both mouse and human platelets. PKCdelta is robustly expressed in human platelets and undergoes transient tyrosine phosphorylation upon stimulation by thrombin or the collagen receptor, GPVI, which becomes sustained in the presence of the pan-PKC inhibitor, Ro 31-8220.

Dynamic tyrosine kinase-regulated signaling and actin polymerisation mediate aggregate stability under shear.

Objective: Aggregate formation on collagen at arteriolar rates of shear is mediated by coordinated signaling between tyrosine kinase-linked and G protein-coupled receptors. We have investigated the role of these receptors and the actin cytoskeleton in maintaining aggregate stability under shear.

Methods And Results: Platelet aggregates are rapidly formed when blood is flowed over collagen at 1000 s(-1) and remain stable over 20 minutes.

Critical role of FcR gamma-chain, LAT, PLCgamma2 and thrombin in arteriolar thrombus formation upon mild, laser-induced endothelial injury in vivo.

Objective: The role of collagen receptor complex GPVI-FcR gamma-chain, PLCgamma2 and LAT in laser-induced thrombosis is unclear. Controversy surrounds whether collagen is exposed in this model or whether thrombosis is dependent on thrombin. This study hypothesized that collagen exposure plays a critical role in thrombus formation in this model, which was tested by investigating contributions of FcR gamma-chain, LAT, PLCgamma2 and thrombin.

Studies on the actin-binding protein HS1 in platelets.

Background: The platelet cytoskeleton mediates the dramatic change in platelet morphology that takes place upon activation and stabilizes thrombus formation. The Arp2/3 complex plays a vital role in these processes, providing the protrusive force for lamellipodia formation. The Arp2/3 complex is highly regulated by a number of actin-binding proteins including the haematopoietic-specific protein HS1 and its homologue cortactin.

Segregation of platelet aggregatory and procoagulant microdomains in thrombus formation: regulation by transient integrin activation.

Objective: Platelets play a dual role in thrombosis by forming aggregates and stimulating coagulation. We investigated the commitment of platelets to these separate functions during collagen-induced thrombus formation in vitro and in vivo.

Methods And Results: High-resolution 2-photon fluorescence microscopy revealed that in thrombus formation under flow, fibrin(ogen)-binding platelets assembled into separate aggregates, whereas distinct patches of nonaggregated platelets exposed phosphatidylserine.

Minimal regulation of platelet activity by PECAM-1.

PECAM-1 is a member of the superfamily of immunoglobulins (Ig) and is expressed on platelets at moderate level. PECAM-1 has been reported to have contrasting effects on platelet activation by the collagen receptor GPVI and the integrin, alphaIIbbeta3, even though both receptors signal through Src-kinase regulation of PLCgamma2. The present study compares the role of PECAM-1 on platelet activation by these two receptors and by the lectin receptor, CLEC-2, which also signals via PLCgamma2.

The glycoprotein VI-phospholipase Cgamma2 signaling pathway controls thrombus formation induced by collagen and tissue factor in vitro and in vivo.

Objective: Both collagen and tissue factor can be initiating factors in thrombus formation. We investigated the signaling pathway of collagen-induced platelet activation in interaction with tissue factor-triggered coagulation during the thrombus-forming process.

Methods And Results: In murine blood flowing over collagen, platelet exposure of phosphatidylserine and procoagulant activity, but not adhesion, completely relied on each of the following signaling modules: glycoprotein VI (GPVI), FcR gamma-chain, Src kinases, adaptor protein LAT, and phospholipase Cgamma2 (PLCgamma2).

Rac1 is essential for platelet lamellipodia formation and aggregate stability under flow.

The role of Rac family proteins in platelet spreading on matrix proteins under static and flow conditions has been investigated by using Rac-deficient platelets. Murine platelets form filopodia and undergo limited spreading on fibrinogen independent of Rac1 and Rac2. In the presence of thrombin, marked lamellipodia formation is observed on fibrinogen, which is abrogated in the absence of Rac1.

Role of the p110delta PI 3-kinase in integrin and ITAM receptor signalling in platelets.

We have investigated the function of the p110delta catalytic subunit of phosphoinositide 3-kinase (PI 3-kinase) in platelets using p110delta knock-out (p110delta(-/-)) mice and p110delta knock-in (p110delta(D910A/D910A)) mice, which express a catalytically inactive form of the enzyme. Aggregation to threshold concentrations of the GPVI-specific agonist, CRP, was partially reduced in p110delta(-/-) and p110delta(D910A/D910A) platelets. This inhibition was overcome by higher concentrations of CRP.

Adhesion of human and mouse platelets to collagen under shear: a unifying model.

There is presently confusion as to the roles of alpha2beta1 and GPVI in supporting platelet adhesion and aggregate formation on collagen at intermediate/high shear. Recent studies have reported essential, partial, or dispensable roles for either receptor in supporting these events, and the possibility that there may be fundamental differences between their roles in human and mouse platelets has been proposed. Further, the recent recognition that Src family tyrosine kinases contribute to signaling by alpha2beta1 and other adhesive receptors, in addition to GPVI, has added to this debate.

GPIb-dependent platelet activation is dependent on Src kinases but not MAP kinase or cGMP-dependent kinase.

Glycoprotein Ib-IX-V (GPIb-IX-V) mediates platelet tethering to von Willebrand factor (VWF), recruiting platelets into the thrombus, and activates integrin alphaIIbbeta3 through a pathway that is dependent on Src kinases. In addition, recent reports indicate that activation of alphaIIbbeta3 by VWF is dependent on protein kinase G (PKG) and mitogen-activated protein (MAP) kinases. The present study compares the importance of these signaling pathways in the activation of alphaIIbbeta3 by GPIb-IX-V.

Glycoproteins VI and Ib-IX-V stimulate tyrosine phosphorylation of tyrosine kinase Syk and phospholipase Cgamma2 at distinct sites.

Glycoproteins GPVI and GPIb-IX-V stimulate robust tyrosine phosphorylation of Syk and PLCg2 (phospholipase Cg2) in washed platelets, but only the former stimulates pronounced activation of phospholipase. Using phospho-specific antibodies, we demonstrate that GPVI, but not GPIb-IX-V, stimulates significant tyrosine phosphorylation of Syk at the autophosphorylation site pY525/526, a marker of Syk activity. In addition, GPVI stimulates tyrosine phosphorylation of PLCg2 at Tyr753 and Tyr759, whereas GPIb-IX-V only induces significant phosphorylation at Tyr753.