A platelet tetraspanin superfamily member,CD151, is required for regulation of thrombus growth and stability in vivo

Summary. Background: This study was designed to determine the role of CD151 in platelet thrombus formation in vivo and define the contribution of platelet vs. endothelial CD151 in regulating platelet thrombus formation in vivo. Methods and Results: Using intravital microscopy and ferric chloride (FeCl₃) injury of mesenteric arterioles, we found that thrombi formed in CD151^+/? and CD151^?/? mice were smaller and less stable, than those formed in CD151^+/+ mice, with a tendency for embolization. Similarly, in Folt’s FeCl₃?induced carotid injury model, both CD151^+/? and CD151^?/? mice showed more prolonged times to 95% vessel occlusion than CD151^+/+ mice. In addition, laser‐induced injury of cremaster muscle arterioles showed that thrombi formed in CD151^+/? and CD151^?/? mice were smaller and less stable than those formed in CD151^+/+ mice. Following platelet depletion/reconstitution with ex vivo‐labeled donor platelets, platelet‐depleted CD151^+/+ mice that received reconstitution with CD151^?/? platelets had smaller thrombi that were unstable and embolized. In contrast, platelet‐depleted CD151^?/? mice that received reconstitution with CD151^+/+ platelets had normal thrombi that were stable. Conclusions: These data provide evidence that platelet CD151 is required for regulating thrombus formation in vivo.     

Diannexin,an annexin A5 homodimer,binds phosphatidylserine with high affinity and is a potent inhibitor of platelet‐mediated events during thrombus formation

Background: Shielding of procoagulant phosphatidylserine (PS) with annexin A5 attenuates thrombosis, but annexin A5 (35.7 kDa) is rapidly cleared from the circulation. In contrast, Diannexin, a 73.1 kDa homodimer of annexin A5, has an extended half‐life. Objectives: To quantify the affinity of Diannexin for PS, examine its interaction with activated platelets and determine its effects on platelet‐mediated events during thrombus formation. Methods: The affinities of Diannexin and annexin A5 for PS‐containing lipid bilayers were compared using surface plasmon resonance, and binding to activated platelets was assessed by flow cytometry. Calibrated automated thrombography and thromboelastography were employed to study the effects of Diannexin on thrombin generation and platelet‐fibrin clot formation, respectively, whereas intravital videomicroscopy was used to examine its effect on platelet accumulation and activation after laser‐induced injury to murine cremaster arterioles, and a tail tip bleeding model was used to explore its effects on hemostasis. Results: Diannexin and annexin A5 bind PS with K_D values of 0.6 and 5 nm , respectively, and both bind to the same subpopulation of PS‐exposing platelets. Diannexin inhibited thrombin generation and platelet‐fibrin clot formation in vitro at 10 nm (P < 0.05–0.001 compared with control), and reduced platelet accumulation at 1 μg g^?1 (P < 0.05) and activation at 0.25 μg g^?1 (P < 0.001) in experimentally induced arterial thrombi in mice while increasing blood loss at 1 μg g^?1 (P < 0.01). Conclusions: Diannexin binds to PS with high affinity and is a potent inhibitor of platelet‐mediated events during thrombus formation.     

Collagen‐mimetic peptides mediate flow‐dependent thrombus formation by high‐ or low‐affinity binding of integrin α2β1 and glycoprotein VI

Summary. Background: Collagen acts as a potent surface for platelet adhesion and thrombus formation under conditions of blood flow. Studies using collagen‐derived triple‐helical peptides have identified the GXX’GER motif as an adhesive ligand for platelet integrin α2β1, and (GPO)_n as a binding sequence for the signaling collagen receptor, glycoprotein VI (GPVI). Objective: The potency was investigated of triple‐helical peptides, consisting of GXX’GER sequences within (GPO)_n or (GPP)_n motifs, to support flow‐dependent thrombus formation. Results: At a high‐shear rate, immobilized peptides containing both the high‐affinity α2β1‐binding motif GFOGER and the (GPO)_n motif supported platelet aggregation and procoagulant activity, even in the absence of von Willebrand factor (VWF). With peptides containing only one of these motifs, co‐immobilized VWF was needed for thrombus formation. The (GPO)_n but not the (GPP)_n sequence induced GPVI‐dependent platelet aggregation and procoagulant activity. Peptides with intermediate affinity (GLSGER, GMOGER) or low‐affinity (GASGER, GAOGER) α2β1‐binding motifs formed procoagulant thrombi only if both (GPO)_n and VWF were present. At a low‐shear rate, immobilized peptides with high‐ or low‐affinity α2β1‐binding motifs mediated formation of thrombi with procoagulant platelets only in combination with (GPO)_n. Conclusions: Triple‐helical peptides with specific receptor‐binding motifs mimic the properties of native collagen I in thrombus formation by binding to both platelet collagen receptors. At a high‐shear rate, either GPIb or high‐affinity (but not low‐affinity) GXX’GER mediates GPVI‐dependent formation of procoagulant thrombi. By extension, high‐affinity binding for α2β1 can control the overall platelet‐adhesive activity of native collagens.     

Disturbed blood flow induces erosive injury to smooth muscle cell‐rich neointima and promotes thrombus formation in rabbit femoral arteries

Summary. Background: Plaque erosion is a cause of atherothrombosis that preferentially occurs on smooth muscle cell (SMC)‐ and proteoglycan‐rich rather than lipid‐rich plaques. However, its underlying mechanisms remain unknown. Objective: To determine whether disturbed blood flow induces erosive injury and thrombus formation on SMC‐rich neointima. Methods: Three weeks after balloon injury, SMC‐rich neointima with increased tissue factor (TF) activity developed in rabbit femoral arteries that were narrowed with a vascular occluder to disturb blood flow after stenosis. Neointimal injury and thrombus formation were assessed at 15, 30, and 180 min after the vascular narrowing. Results: Endothelial detachment, platelet adhesion and neointimal cell apoptosis became evident at the post‐stenotic regions of all femoral arteries (n = 5) within 15 min of narrowing. Mural thrombi composed of platelet and fibrin developed after 30 min, and then occlusive thrombi were generated in three out of five vessels after 180 min. The identical vascular narrowing of normal femoral arteries also induced endothelial detachment with small platelet thrombi at post‐stenotic regions, but fibrin and occlusive thrombi did not develop. Computational simulation analysis indicated that oscillatory shear stress contributes to the development of erosive damage to the neointima. Conclusions: These results suggest that disturbed post‐stenotic blood flow can induce erosive injury in SMC‐rich plaques and promote thrombus formation that results in vascular events.     

Laminin promotes coagulation and thrombus formation in a factor XII‐dependent manner

Summary. Background: Laminin is the most abundant non‐collagenous protein in the basement membrane. Recent studies have shown that laminin supports platelet adhesion, activation and aggregation under flow conditions, highlighting a possible role for laminin in hemostasis. Objective: To investigate the ability of laminin to promote coagulation and support thrombus formation under shear. Results and methods: Soluble laminin accelerated factor (F) XII activation in a purified system, and shortened the clotting time of recalcified plasma in a FXI‐ and FXII‐dependent manner. Laminin promoted phosphatidylserine exposure on platelets and supported platelet adhesion and fibrin formation in recalcified blood under shear flow conditions. Fibrin formation in laminin‐coated capillaries was abrogated by an antibody that interferes with FXI activation by activated FXII, or an antibody that blocks activated FXI activation of FIX. Conclusion: This study identifies a role for laminin in the initiation of coagulation and the formation of platelet‐rich thrombi under shear conditions in a FXII‐dependent manner.     

TC21/RRas2 regulates glycoprotein VI–FcRγ‐mediated platelet activation and thrombus stability

Essentials

• RAS proteins are expressed in platelets but their functions are largely uncharacterized.
• TC21/RRas2 is required for glycoprotein VI‐induced platelet responses and for thrombus stability in vivo.
• TC21 regulates platelet aggregation by control of α_IIbβ₃ integrin activation, via crosstalk with Rap1b.
• This is the first indication of functional importance of a proto‐oncogenic RAS protein in platelets.

Summary

Background

Many RAS family small GTPases are expressed in platelets, including RAC, RHOA, RAP, and HRAS/NRAS/RRAS1, but most of their signaling and cellular functions remain poorly understood. Like RRAS1, TC21/RRAS2 reverses HRAS‐induced suppression of integrin activation in CHO cells. However, a role for TC21 in platelets has not been explored.

Objectives

To determine TC21 expression in platelets, TC21 activation in response to platelet agonists, and roles of TC21 in platelet function in in vitro and in vivo thrombosis.

Results

We demonstrate that TC21 is expressed in human and murine platelets, and is activated in response to agonists for the glycoprotein (GP) VI–FcRγ immunoreceptor tyrosine‐based activation motif (ITAM)‐containing collagen receptor, in an Src‐dependent manner. GPVI‐induced platelet aggregation, integrin α_IIbβ₃ activation, and α‐granule and dense granule secretion, as well as phosphorylation of Syk, phospholipase Cγ2, AKT, and extracellular signal‐regulated kinase, were inhibited in TC21‐deficient platelets ex vivo. In contrast, these responses were normal in TC21‐deficient platelets following stimulation with P2Y, protease‐activated receptor 4 and C‐type lectin receptor 2 receptor agonists, indicating that the function of TC21 in platelets is GPVI–FcRγ‐ITAM‐specific. TC21 was required for GPVI‐induced activation of Rap1b. TC21‐deficient mice did not show a significant delay in injury‐induced thrombosis as compared with wild‐type controls; however, thrombi were unstable. Hemostatic responses showed similar effects.

Conclusions

TC21 is essential for GPVI–FcRγ‐mediated platelet activation and for thrombus stability in vivo via control of Rap1b and integrins.

     

Protein kinase C alpha and beta are positive regulators of thrombus formation in vivo in a zebrafish (Danio rerio) model of thrombosis

Summary. Background: The zebrafish (Danio rerio) is becoming an attractive model organism for the assessment of gene function in thrombosis in vivo. Zebrafish, as a thrombosis model, have several advantages, with the capacity to follow thrombus formation at high resolution in real time using intravital microscopy, without the need for complex surgical techniques, and the capability to rapidly knockdown gene expression using morpholino antisense approaches. Objectives: We have recently shown, in mouse models, that protein kinase C alpha (PKCα) plays a critical role in regulating thrombus formation in vivo. PKC beta (β) plays a non‐redundant role also in platelet function in vitro, but the function of this gene had not yet been assessed in vivo. Methods: In the present study, we analyzed the function of both PKCα and PKCβ in the zebrafish model in vivo, by live imaging using a laser‐induced injury of the main caudal artery in 3‐day‐old larvae. Results: We showed that D. rerio express orthologs of both the PKCα and PKCβ genes, with high sequence identity. Translation blocking and splice‐blocking morpholinos effectively and specifically knockdown expression of these genes and knockdown with either morpholino leads to attenuated thrombus formation, as assessed by several quantitative parameters including time to initial adhesion and peak thrombus surface area. Conclusions: Our data indicate that these two highly related genes play non‐redundant roles in regulating thrombosis, an observation that supports our previous in vitro murine data, and suggests unique roles, and possibly unique regulation, for PKCα and PKCβ in controlling platelet function in vivo.     

4‐Thio‐deoxyuridylate‐modified thrombin aptamer and its inhibitory effect on fibrin clot formation,platelet aggregation and thrombus growth on subendothelial matrix

Summary. Background: The consensus thrombin aptamer C15‐mer is a single‐stranded DNA of 15 nucleotides [d(GGTTGGTGTGGTTGG)] that was identified by the selection of thrombin‐binding molecules from a large combinatorial library of oligonucleotides. It is capable of inhibiting thrombin at nanomolar concentrations through binding to a specific region within thrombin exosite 1. As has been shown in our earlier studies, the 4‐thio‐deoxyuridylate (s4dU)‐containing oligonucleotides have high affinity for a number of proteins, due to the reduced hydrophilic character of the modified oligonucleotide. Methods: Three different analogs of the original thrombin‐inhibiting sequence, in which some of the thymidylate residues were replaced by 4‐thio‐deoxyuridylates, were synthesized. The inhibitory effect of modified aptamers was tested on thrombin‐catalyzed fibrin clot formation and fibrinopeptide A release from fibrinogen, thrombin‐induced platelet aggregation/secretion, and the formation of thrombus on coverslips coated with human collagen type III, thrombin‐treated fibrinogen or subendothelial matrix of human microvascular endothelial cells. Results: As compared with the C15‐mer, the analog with the sequence GG(s4dU)TGG(s4dU)G(s4dU)GGT(s4dU)GG (UC15‐mer) showed a 2‐fold increased inhibition of thrombin‐catalyzed fibrin clot formation, fibrinopeptide A release, platelet aggregation and secretion in human plasma and thrombus formation on thrombin‐treated fibrinogen surfaces under flow conditions. Concerning the inhibition of thrombin‐induced fibrin formation from purified fibrinogen and activation of washed platelets, UC15‐mer was 3‐fold and twelve‐fold more effective than C15‐mer, respectively. Conclusion: The replacement of four thymidylate residues in C15‐mer by 4‐thio‐deoxyuridylate resulted in a new thrombin aptamer with increased anticoagulant and antithrombotic properties.     

Rap1b is critical for glycoprotein VI-mediated but not ADP receptor-mediated α2β1 activation

Summary. Background: The platelet α₂β₁ integrin functions as both an adhesion and signaling receptor upon exposure to collagen. Recent studies have indicated that α₂β₁ function can be activated via inside‐out signaling, similar to the prototypical platelet integrin α_IIbβ₃. However, signaling molecules that regulate α₂β₁ activation in platelets are not well defined. A strong candidate molecule is the small GTPase Rap1b, the dominant platelet isoform of Rap1, which regulates α_IIbβ₃ activation. Objectives: We hypothesized that Rap1b positively regulates α₂β₁ during agonist‐induced platelet activation. Methods: To test whether Rap1b activates α₂β₁ downstream of glycoprotein (GP)VI or other platelet receptors, we stimulated platelets purified from Rap1b^?/? or wild‐type mice with diverse agonists and measured α₂β₁ activation using fluorescein isothiocyanate‐labeled monomeric collagen. We also examined the role of Rap1b in outside‐in signaling pathways by analyzing adhesion and spreading of Rap1b^?/? or wild‐type platelets on monomeric, immobilized collagen. Finally, we monitored the activation status of related Rap GTPases to detect changes in signaling pathways potentially associated with Rap1b‐mediated events. Results: Rap1b^?/? platelets displayed comparable ADP‐induced or thrombin‐induced α₂β₁ activation as wild‐type platelets, but reduced convulxin‐dependent α₂β₁ activation. Rap1b^?/? platelets exhibited increased spreading on immobilized collagen but similar adhesion to immobilized collagen compared to wild‐type platelets. Rap1b^?/? platelets also showed Rap1a and Rap2 activation upon agonist stimulation, possibly revealing functional compensation among Rap family members. Conclusions: Rap1b is required for maximal GPVI‐induced but not ADP‐induced activation of α₂β₁ in murine platelets.     

Redundant functions of phospholipases D1 and D2 in platelet α‐granule release

Summary. Background: Platelet activation and aggregation are crucial for primary hemostasis, but can also result in occlusive thrombus formation. Agonist‐induced platelet activation involves different signaling pathways leading to the activation of phospholipases, which produce second messengers. The role of phospholipase C (PLC) in platelet activation is well established, but less is known about the relevance of phospholipase D (PLD) . Objective and methods: The aim of this study was to determine a potential function of PLD2 in platelet physiology. Thus, we investigated the function of PLD2 in platelet signaling and thrombus formation, by generating mice lacking PLD2 or both PLD1 and PLD2. Adhesion, activation and aggregation of PLD‐deficient platelets were analyzed in vitro and in vivo. Results: Whereas the absence of PLD2 resulted in reduced PLD activity in platelets, it had no detectable effect on the function of the cells in vitro and in vivo. However, the combined deficiency of both PLD isoforms resulted in defective α‐granule release and protection in a model of FeCl₃‐induced arteriolar thrombosis, effects that were not observed in mice lacking only one PLD isoform. Conclusion: These results reveal redundant roles of PLD1 and PLD2 in platelet α‐granule secretion, and indicate that this may be relevant for pathologic thrombus formation.     

Shiga toxin binds to activated platelets

Summary. Hemolytic uremic syndrome (HUS) is associated with acute renal failure in children and can be caused by Shiga toxin (Stx)‐producing Escherichia coli. Thrombocytopenia and formation of renal thrombi are characteristic of HUS, suggesting that platelet activation is involved in its pathogenesis. However, whether Shiga toxin directly activates platelets is controversial. The present study evaluates if potential platelet sensitization during isolation by different procedures influences platelet interaction with Shiga toxin. Platelets isolated from sodium citrate anticoagulated blood were exposed during washing to EDTA and higher g forces than platelets prepared from acid‐citrate‐dextrose (ACD) plasma. Platelet binding of Stx was significantly higher in EDTA‐washed preparations relative to ACD‐derived platelets. Binding of Stx was also increased with ACD‐derived platelets when activated with thrombin (1 U mL^?1) and exposure of the Gb3 Stx receptor was detected only on platelets subjected to EDTA, higher g forces or thrombin. EDTA‐exposed platelets lost their normal discoid shape and were larger. P‐selectin (CD62P) exposure was significantly increased in EDTA‐washed preparations relative to ACD‐derived platelets, suggesting platelet activation. Taken together, these results suggest that direct binding of Stx occurs only on ‘activated’ platelets rather than on resting platelets. The ability of Stx to interact with previously activated platelets may be an important element in understanding the pathogenesis of HUS.     

Extracellular protein disulfide isomerase regulates feedback activation of platelet thrombin generation via modulation of coagulation factor binding

Summary. Background: Protein disulfide isomerase (PDI) controls platelet integrin function, tissue‐factor (TF) activation, and concentrates at fibrin and thrombus formation sites of vascular injury. Objective: To investigate the involvement of surface thiol isomerases and especially PDI, in thrombin‐mediated thrombin amplification on human platelets. Methods/results: Using a newly developed thrombin‐dependent platelet thrombin generation assay, we observed that the feedback activation of thrombin generation on the platelet surface does not depend on TF, as anti‐TF antibodies inhibiting TF‐induced thrombin formation in platelet‐depleted plasma had no effect compared with vehicle‐treated controls. Feedback activation of thrombin generation in the presence of platelets was significantly diminished by membrane impermeant thiol blockers or by the thiol isomerase‐inhibitors bacitracin and anti‐PDI antibody RL90, respectively. Platelet thrombin formation depends on binding of coagulation factors to the platelet surface. Therefore, involvement of thiol isomerases in this binding was investigated. As shown by confocal microscopy and flow cytometry, thrombin‐stimulated platelets exhibited increased surface‐associated PDI as well as extracellular disulfide reductase activity compared with unstimulated platelets. Flow cytometric analysis revealed that membrane impermeant thiol blockers or PDI inhibitors, which had been added after platelet stimulation and after phosphatidylserine exposure to exclude their influence on primary platelet activation, significantly inhibited binding of all coagulation factors to thrombin‐stimulated platelets. Conclusions: Thus, surface‐associated PDI is an important regulator of coagulation factor ligation to thrombin‐stimulated platelets and of subsequent feedback activation of platelet thrombin generation. Cell surface thiol isomerases might be therefore powerful targets to control hemostasis and thrombosis.     

The role of thrombin activatable fibrinolysis inhibitor and factor XI in platelet‐mediated fibrinolysis resistance: a thromboelastographic study in whole blood

Summary. Background: The resistance of platelet‐rich thrombi to fibrinolysis is generally attributed to clot retraction and platelet PAI‐1 release. The role of TAFI in platelet‐mediated resistance to lysis is unclear. Objective: We investigated the contribution of TAFI to the antifibrinolytic effect of platelets in whole blood by thromboelastography. Methods: Platelet‐poor (PP‐WB, < 40 × 10³ μL^?1) and platelet‐rich (PR‐WB, > 400 × 10³ μL^?1) blood samples were obtained from normal human blood (N‐WB, 150–220 × 10³ μL^?1). Clot lysis time was measured by thromboelastography in recalcified blood supplemented with t‐PA (100 ng mL^?1) and tissue factor (1:1000 Recombiplastin). Results: t‐PA‐induced lysis time increased in parallel with platelet concentration (up to 3‐fold). Neutralization of TAFI, but not of PAI‐1, shortened the lysis time by ～ 50% in PR‐WB and by < 10% in PP‐WB. Accordingly, prothrombin F1+2 and TAFIa accumulation was greater in PR‐WB than in PP‐WB. A similar TAFI‐dependent inhibition of fibrinolysis was observed when clot retraction was prevented by cytochalasin D or abciximab, or when platelet membranes were tested. Moreover, in blood with an intact contact system, platelet‐mediated fibrinolysis resistance was attenuated by an anti‐FXI but not by an anti F‐XII antibody. Finally, platelets made the clots resistant to the profibrinolytic effect of heparin concentrations displaying a strong anticoagulant activity. Conclusions: Our data indicate that TAFI activation is one major mechanism whereby platelets make clots resistant to fibrinolysis and underscore the importance of TAFI inhibitors as new antithrombotic agents.     

EMMPRIN (CD147/basigin) mediates platelet–monocyte interactions in vivo and augments monocyte recruitment to the vascular wall

Summary. Background: Platelets play a central role in hemostasis, in inflammatory diseases such as atherosclerosis, and during thrombus formation following vascular injury. Thereby, platelets interact intensively with monocytes and enhance their recruitment to the vascular wall. Objectives: To investigate the role of the extracellular matrix metalloproteinase inducer (EMMPRIN) in platelet–monocyte interactions. Methods and Results: Isolated human monocytes were perfused in vitro over firmly adherent platelets to allow investigation of the role of EMMPRIN in platelet–monocyte interactions under flow conditions. Monocytes readily bound to surface‐adherent platelets. Both antibody blockade and gene silencing of monocyte EMMPRIN substantially attenuated firm adhesion of monocytes to platelets at arterial and venous shear rates. In vivo, platelet interactions with the murine monocyte cell line ANA‐1 were significantly decreased when ANA‐1 cells were pretreated with EMMPRIN‐silencing small interfering RNA prior to injection into wild‐type mice. Using intravital microscopy, we showed that recruitment of EMMPRIN‐silenced ANA‐1 to the injured carotid artery was significantly reduced as compared with control cells. Further silencing of EMMPRIN resulted in significantly fewer ANA‐1–platelet aggregates in the mouse circulation as determined by flow cytometry. Finally, we identified glycoprotein (GP)VI as a critical corresponding receptor on platelets that mediates interaction with monocyte EMMPRIN. Thus, blocking of GPVI inhibited the effect of EMMPRIN on firm monocyte adhesion to platelets under arterial flow conditions in vitro, and abrogated EMMPRIN‐mediated platelet–monocyte aggregate formation in vivo. Conclusions: EMMPRIN supports platelet–monocyte interactions and promotes monocyte recruitment to the arterial wall. Therefore, EMMPRIN might represent a novel target to reduce vascular inflammation and atherosclerotic lesion development.     

Signaling role of CD36 in platelet activation and thrombus formation on immobilized thrombospondin or oxidized low‐density lipoprotein

Summary. Background and Objective: Platelets abundantly express glycoprotein CD36 with thrombospondin‐1 (TSP1) and oxidized low‐density lipoprotein (oxLDL) as proposed ligands. How these agents promote platelet activation is still poorly understood. Methods and Results: Both TSP1 and oxLDL caused limited activation of platelets in suspension. However, immobilized TSP1 and oxLDL, but not LDL, strongly supported platelet adhesion and spreading with a major role of CD36. Platelet spreading was accompanied by potent Ca²⁺ rises, and resulted in exposure of P‐selectin and integrin activation, all in a CD36‐dependent manner with additional contributions of α_IIbβ₃ and ADP receptor stimulation. Signaling responses via CD36 involved activation of the protein tyrosine kinase Syk. In whole blood perfusion, co‐coating of TSP1 or oxLDL with collagen enhanced thrombus formation at high‐shear flow conditions, with increased expression on platelets of activated α_IIbβ₃, P‐selectin and phosphatidylserine, again in a CD36‐dependent way. Conclusions: Immobilized TSP1 and oxLDL activate platelets partly via CD36 through a Syk kinase‐dependent Ca²⁺ signaling mechanism, which enhances collagen‐dependent thrombus formation under flow. These findings provide novel insight into the role of CD36 in hemostasis.     

Coronary artery bypass graft surgery up‐regulates genes involved in platelet aggregation

Summary. Background: During and shortly after coronary artery bypass graft (CABG) surgery, there is an increase in thromboembolic events. CABG, a strong inflammatory stimulus, is associated with a hypercoaguable state. Platelets might contribute to this hypercoaguable state because they have a pivotal role in thrombosis. In the days following surgery there is augmented platelet regeneration in response to the inflammatory stimulus. Objectives: The aim of this study was to investigate any changes in platelet mRNA profiles to test the hypothesis that post‐CABG surgery platelets are associated with a prothrombotic state. Methods: Blood was sampled and platelets purified from 11 patients before and 3–6 days after CABG. Gene expression profiling was performed using low density array (LDA) plates for seven of the patients. Results: Forty‐five genes were examined and those significantly up‐regulated were glycoprotein (GP)IIb, GPIIIa and cyclooxygenase‐1 (COX‐1). These findings were confirmed in four more patients, including flow cytometry analysis of the GPIIb/IIIa receptor. Conclusions: CABG surgery up‐regulates mRNA and protein levels of proteins that are key players in platelet aggregation. Marked elevation of GPIIb/IIIa mRNA levels results in significantly increased GPIIb/IIIa expression in platelets post‐CABG surgery, which may be a reason for increased thrombus formation and myocardial infarction after CABG.     

Non‐genomic effects of PPARγ ligands: inhibition of GPVI‐stimulated platelet activation

Summary. Background: Peroxisome proliferator‐activated receptor‐_γ (PPAR_γ) is expressed in human platelets although in the absence of genomic regulation in these cells, its functions are unclear. Objective: In the present study, we aimed to demonstrate the ability of PPAR_γ ligands to modulate collagen‐stimulated platelet function and suppress activation of the glycoprotein VI (GPVI) signaling pathway. Methods: Washed platelets were stimulated with PPAR_γ ligands in the presence and absence of PPAR_γ antagonist GW9662 and collagen‐induced aggregation was measured using optical aggregometry. Calcium levels were measured by spectrofluorimetry in Fura‐2AM‐loaded platelets and tyrosine phosphorylation levels of receptor‐proximal components of the GPVI signaling pathway were measured using immunoblot analysis. The role of PPAR_γ agonists in thrombus formation was assessed using an in vitro model of thrombus formation under arterial flow conditions. Results: PPAR_γ ligands inhibited collagen‐stimulated platelet aggregation that was accompanied by a reduction in intracellular calcium mobilization and P‐selectin exposure. PPAR_γ ligands inhibited thrombus formation under arterial flow conditions. The incorporation of GW9662 reversed the inhibitory actions of PPAR_γ agonists, implicating PPAR_γ in the effects observed. Furthermore, PPAR_γ ligands were found to inhibit tyrosine phosphorylation levels of multiple components of the GPVI signaling pathway. PPAR_γ was found to associate with Syk and LAT after platelet activation. This association was prevented by PPAR_γ agonists, indicating a potential mechanism for PPAR_γ function in collagen‐stimulated platelet activation. Conclusions: PPAR_γ agonists inhibit the activation of collagen‐stimulation of platelet function through modulation of early GPVI signalling.     

Platelet-derived NO slows thrombus growth on a collagen type III surface

Nitric oxide (NO) is a free radical that plays an important role in modulating platelet adhesion and aggregation. Platelets are a source of vascular NO, but since erythrocytes avidly scavenge NO, the functional significance of platelet-derived NO is not clear. Our purpose was to determine if NO from platelets affects platelet thrombus formation in the presence of anticoagulated whole blood in an in vitro parallel plate flow system. We studied platelet adhesion and aggregation on a collagen type III surface in the presence of physiologically relevant fluid mechanical shear stress. We found that certain receptor mediated agonists (insulin and isoproterenol) caused a concentration dependent reduction in thrombus formation at a shear rate of 1000 s^-1. This effect was mediated by NO since it was abolished in the presence of the NO inhibitor L-nitro-arginine-methyl-ester (L-NAME). As expected, at venous levels of shear rate (100 s^-1) neither of the agonists had any effect on thrombus formation since platelet adhesion does not depend on activation at these low levels of shear. Interestingly, at a shear rate of 2000 s^-1 the addition of L-NAME caused an increase in platelet coverage suggesting that shear, by itself, induces NO production by platelets. This is the first demonstration of shear stress causing platelets to produce an inhibitor of platelet activation. These results demonstrate that the development of a platelet thrombus is regulated in a complex way and that platelets produce functionally significant amounts of NO even in the presence of whole blood.     

Impaired platelet prostacyclin receptor activity: a monozygotic twin study discordant for spinal cord injury

Coronary artery disease (CAD) has been reported to occur prematurely in individuals with spinal cord injury (SCI). Although persons with SCI have metabolic abnormalities that may predispose them to CAD, other potential aetiologies may also be operative. Increased platelet aggregation, among other factors, initiates thrombus formation at the site of the vessel injury, which may acutely obstruct arterial blood flow. Prostacyclin is known to have a beneficial effect to inhibit platelet aggregation and prevent thrombus formation. Platelets were studied from 12 pairs of monozygotic twins, one co‐twin with SCI. Each twin pair had similar patterns of platelet aggregation with adenosine diphosphate (ADP), thrombin or collagen, as well as inhibition of platelet aggregation by prostacyclin (PGE₁/I₂) and synthesis of cyclic adenosine mono phosphate (AMP) by the prostanoid. However, the twin pairs differed in their response to PGE₁/I₂ inhibition of platelet‐stimulated thrombin generation that was completely inhibited in non‐SCI platelets but not in SCI platelets. Scatchard analysis of the binding of ³H‐prostaglandin E₁, a stable prostacyclin receptor probe, showed the presence of one high‐affinity (K_d1=8·1 ± 2·8 nM ; n_l=168 ± 35 sites per platelet) and one low‐affinity (K_d2=1·1 ± 0·22 μM ; n₂=1772 ± 220 sites per cell) prostacyclin receptor in normal platelets, whereas in SCI platelets there was a significant loss (P<0·00l) of high‐affinity receptor sites (K_d1=6·34 ± 1·80 nM ; n₁=42 ± 11 sites per platelet) with no significant change in the low‐affinity receptor sites (K_d2=1·2 ± 0·23 μM ; n₂=1860 ± 412 sites per cell). These discordant platelet findings in identical twin pairs raises the possibility of an environmental aetiology for accelerated CAD in those with SCI. The loss of inhibitory effect of PGI₂ on thrombin generation in the twin with SCI appears to be because of loss of platelet high‐affinity prostanoid receptors, which may contribute to atherogenesis in individuals with SCI.