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1.
Blood ; 107(11): 4346-53, 2006 Jun 01.
Article in English | MEDLINE | ID: mdl-16391010

ABSTRACT

Fibrillar collagens are among the most potent activators of platelets and play an important role in the initiation of thrombosis. The glycoprotein VI (GPVI)/FcRgamma-chain complex is a central collagen receptor and inhibitors of GPVI produce a major defect in arterial thrombogenesis. In this study we have examined arterial thrombus formation in mice lacking the GPVI/FcRgamma-chain complex (FcRgamma(-/-)). Using 3 distinct arterial thrombosis models involving deep vascular injury, we demonstrate that deficiency of GPVI/FcRgamma is not associated with a major defect in arterial thrombus formation. In contrast, with milder vascular injury deficiency of GPVI/FcRgamma was associated with a 30% reduction in thrombus growth. Analysis of FcRgamma(-/-) platelets in vitro, using thrombin-dependent and -independent thrombosis models, demonstrated a major role for thrombin in overcoming the thrombosis defect associated with GPVI/FcRgamma deficiency. Inhibition of thrombin in vivo produced a much greater defect in thrombus formation in mice lacking GPVI/FcRgamma compared with normal controls. Similarly, thrombin inhibition produced a marked prolongation in bleeding time in FcRgamma(-/-) mice relative to wild-type mice. Our studies define an important role for thrombin in overcoming the hemostatic and thrombotic defect associated with GPVI/FcRgamma deficiency. Moreover, they raise the interesting possibility that the full antithrombotic potential of GPVI receptor antagonists may only be realized through the concurrent administration of anticoagulant agents.


Subject(s)
Platelet Membrane Glycoproteins/deficiency , Receptors, IgG/deficiency , Thrombin/physiology , Thrombosis/etiology , Animals , Arterial Occlusive Diseases/etiology , Arterial Occlusive Diseases/prevention & control , Blood Platelets/chemistry , Blood Vessels/injuries , Disease Models, Animal , Hirudins/pharmacology , Mice , Mice, Knockout , Platelet Activation , Thrombin/antagonists & inhibitors , Thrombosis/prevention & control
2.
Nat Med ; 11(5): 507-14, 2005 May.
Article in English | MEDLINE | ID: mdl-15834429

ABSTRACT

Platelet activation at sites of vascular injury is essential for the arrest of bleeding; however, excessive platelet accumulation at regions of atherosclerotic plaque rupture can result in the development of arterial thrombi, precipitating diseases such as acute myocardial infarction and ischemic stroke. Rheological disturbances (high shear stress) have an important role in promoting arterial thrombosis by enhancing the adhesive and signaling function of platelet integrin alpha(IIb)beta(3) (GPIIb-IIIa). In this study we have defined a key role for the Type Ia phosphoinositide 3-kinase (PI3K) p110beta isoform in regulating the formation and stability of integrin alpha(IIb)beta(3) adhesion bonds, necessary for shear activation of platelets. Isoform-selective PI3K p110beta inhibitors have been developed which prevent formation of stable integrin alpha(IIb)beta(3) adhesion contacts, leading to defective platelet thrombus formation. In vivo, these inhibitors eliminate occlusive thrombus formation but do not prolong bleeding time. These studies define PI3K p110beta as an important new target for antithrombotic therapy.


Subject(s)
Arteries/pathology , Phosphatidylinositol 3-Kinases/metabolism , Platelet Adhesiveness/physiology , Platelet Glycoprotein GPIIb-IIIa Complex/metabolism , Signal Transduction/physiology , Thrombosis/metabolism , Animals , Bleeding Time , Blood Platelets/metabolism , Flow Cytometry , Isoenzymes/metabolism , Mice , Mice, Knockout , Phosphoinositide-3 Kinase Inhibitors , Rheology , Serotonin/metabolism , Thrombosis/pathology , rap GTP-Binding Proteins/metabolism
3.
J Biol Chem ; 279(20): 21439-48, 2004 May 14.
Article in English | MEDLINE | ID: mdl-14981081

ABSTRACT

Plasma von Willebrand factor (VWF) is a multimeric glycoprotein from endothelial cells and platelets that mediates adhesion of platelets to sites of vascular injury. In the shear force of flowing blood, however, only the very large VWF multimers are effective in capturing platelets. The multimeric size of VWF can be controlled by proteolysis at the Tyr(842)-Met(843) peptide bond by ADAMTS13 or cleavage of the disulfide bonds that hold VWF multimers together by thrombospondin-1 (TSP-1). The average multimer size of plasma VWF in TSP-1 null mice was significantly smaller than in wild type mice. In addition, the multimer size of VWF released from endothelium in vivo was reduced more rapidly in TSP-1 null mice than in wild type mice. TSP-1, like ADAMTS13, bound to the VWF A3 domain. TSP-1 in the wild type mice, therefore, may compete with ADAMTS13 for interaction with the A3 domain and slow the rate of VWF proteolysis. TSP-1 is stored in platelet alpha-granules and is released upon platelet activation. Significantly, platelet VWF multimer size was reduced upon lysis or activation of wild type murine platelets but not TSP-1 null platelets. This difference had functional consequences in that there was an increase in collagen- and VWF-mediated aggregation of the TSP-1 null platelets under both static and shear conditions. These findings indicate that TSP-1 influences plasma and platelet VWF multimeric size differently and may be more relevant for control of the VWF released from platelets.


Subject(s)
Thrombospondin 1/metabolism , von Willebrand Factor/metabolism , Animals , Blood Platelets/physiology , Dipeptides/chemistry , Mice , Mice, Knockout , Thrombospondin 1/deficiency , Thrombospondin 1/genetics , von Willebrand Factor/chemistry
4.
J Biol Chem ; 278(37): 34812-22, 2003 Sep 12.
Article in English | MEDLINE | ID: mdl-12832405

ABSTRACT

Platelet adhesion to fibrinogen is important for platelet aggregation and thrombus growth. In this study we have examined the mechanisms regulating platelet adhesion on immobilized fibrinogen under static and shear conditions. We demonstrate that integrin alpha IIb beta 3 engagement of immobilized fibrinogen is sufficient to induce an oscillatory calcium response, necessary for lamellipodial formation and platelet spreading. Released ADP increases the proportion of platelets exhibiting a cytosolic calcium response but is not essential for calcium signaling or lamellipodial extension. Pretreating platelets with the Src kinase inhibitor PP2, the inositol 1,4,5-trisphosphate (IP3) receptor antagonist 2-aminoethoxydiphenyl borate (APB-2), or the phospholipase C (PLC) inhibitor U73122 abolished calcium signaling and platelet spreading, suggesting a major role for Src kinase-regulated PLC isoforms in these processes. Analysis of PLC gamma 2-/- mouse platelets revealed a major role for this isoform in regulating cytosolic calcium flux and platelet spreading on fibrinogen. Under flow conditions, platelets derived from PLC gamma 2-/- mice formed less stable adhesive interactions with fibrinogen, particularly in the presence of ADP antagonists. Our studies define an important role for PLC gamma 2 in integrin alpha IIb beta 3-dependent calcium flux, necessary for stable platelet adhesion and spreading on fibrinogen. Furthermore, they establish an important cooperative signaling role for PLC gamma 2 and ADP in regulating platelet adhesion efficiency on fibrinogen.


Subject(s)
Blood Platelets/physiology , Calcium Signaling/physiology , Fibrinogen/physiology , Platelet Adhesiveness/physiology , Platelet Glycoprotein GPIIb-IIIa Complex/physiology , Type C Phospholipases/blood , Animals , Apyrase/pharmacology , Aspirin/pharmacology , Calcium Signaling/drug effects , Humans , Kinetics , Mice , Mice, Knockout , Phospholipase C gamma , Platelet Aggregation Inhibitors/pharmacology , Platelet Glycoprotein GPIIb-IIIa Complex/drug effects , Type C Phospholipases/deficiency
5.
Blood ; 99(1): 151-8, 2002 Jan 01.
Article in English | MEDLINE | ID: mdl-11756165

ABSTRACT

Platelet adhesion and aggregation at sites of vascular injury are critically dependent on the interaction between von Willebrand factor (VWF) and 2 major platelet adhesion receptors, glycoprotein (GP) Ib/V/IX and integrin alpha(IIb)beta(3). GP Ib/V/IX binding to VWF mediates platelet tethering and translocation, whereas activation of integrin alpha(IIb)beta(3) promotes cell arrest. To date, the signaling pathways used by the VWF-GP Ib/V/IX interaction to promote activation of integrin alpha(IIb)beta(3), particularly under shear, have remained poorly defined. In this study, the potential involvement of type 1 phosphoinositide (PI) 3-kinases in this process was investigated. Results show that platelet adhesion and spreading on immobilized VWF results in a specific increase in the PI 3-kinase lipid product, PtdIns(3,4)P(2). Under static conditions, inhibiting PI 3-kinase with LY294002 or wortmannin did not prevent platelet adhesion, integrin alpha(IIb)beta(3) activation, or platelet spreading although it significantly delayed the onset of these events. In contrast, PI 3-kinase inhibition under shear dramatically reduced both platelet adhesion and spreading. Real-time analysis of intracellular calcium demonstrated that under static conditions inhibiting PI 3-kinase delayed the onset of intracellular fluxes in adherent platelets, but did not affect the final magnitude of the calcium response. However, under shear, inhibiting PI 3-kinase dramatically reduced intracellular calcium mobilization and integrin alpha(IIb)beta(3) activation, resulting in impaired thrombus growth. The studies demonstrate a shear-dependent role for PI 3-kinase in promoting platelet adhesion on immobilized VWF. Under static conditions, platelets appear to mobilize intracellular calcium through both PI 3-kinase-dependent and -independent mechanisms, whereas under shear PI 3-kinase is indispensable for VWF-induced calcium release.


Subject(s)
Blood Platelets/physiology , Hemorheology , Phosphatidylinositol 3-Kinases/blood , Platelet Glycoprotein GPIIb-IIIa Complex/physiology , Platelet Glycoprotein GPIb-IX Complex/physiology , Signal Transduction , Androstadienes/pharmacology , Blood Platelets/drug effects , Calcium/blood , Chromones/pharmacology , Enzyme Inhibitors/pharmacology , Humans , Morpholines/pharmacology , Phosphatidylinositol Phosphates/blood , Platelet Adhesiveness , Wortmannin , von Willebrand Factor/metabolism
6.
J Biol Chem ; 277(4): 2965-72, 2002 Jan 25.
Article in English | MEDLINE | ID: mdl-11713259

ABSTRACT

We have investigated the calcium signaling relationship between the two major platelet adhesion receptors, glycoprotein Ib/V/IX (GPIb/V/IX) and integrin alpha(IIb)beta(3), involved in regulating platelet adhesion on von Willebrand factor (vWf) under flow. Our studies demonstrate that GPIb engagement of immobilized vWf elicits a transient calcium spike that may function to promote reversible arrest of translocating platelets. Subsequent integrin alpha(IIb)beta(3) engagement of vWf promotes sustained calcium oscillations that are essential for the maintenance of irreversible adhesion. GPIb-induced calcium spikes appear distinct from those initiated by integrin alpha(IIb)beta(3), in that the former are exclusively mediated through release of intracellular calcium stores via a signaling mechanism independent of PI 3-kinase. In contrast, integrin alpha(IIb)beta(3)-dependent calcium flux involves a PI 3-kinase-dependent signaling mechanism linked to intracellular calcium mobilization and subsequent transmembrane calcium influx. Studies employing the caged calcium chelator (o-nitrophenyl-EGTA) demonstrate that transient calcium spikes initiate a transient phase of platelet arrest that is converted to irreversible adhesion with the development of sustained oscillatory calcium flux. These studies demonstrate the existence of a dual step calcium signaling mechanism utilized by GPIb and integrin alpha(IIb)beta(3) that serves to regulate the dynamics of platelet adhesion under flow.


Subject(s)
Blood Platelets/metabolism , Calcium/metabolism , Platelet Glycoprotein GPIIb-IIIa Complex/metabolism , Platelet Glycoprotein GPIb-IX Complex/metabolism , Cell Adhesion , Chelating Agents/pharmacology , Cytosol/metabolism , Dose-Response Relationship, Drug , Egtazic Acid/chemistry , Egtazic Acid/pharmacology , Humans , Kinetics , Mitogen-Activated Protein Kinases/metabolism , Models, Biological , Phosphatidylinositol 3-Kinases/metabolism , Protein Binding , Protein Transport , Signal Transduction , Time Factors
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