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.     

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.     

A role for adhesion and degranulation‐promoting adapter protein in collagen‐induced platelet activation mediated via integrin α2β1

Summary. Background: Collagen‐induced platelet activation is a key step in the development of arterial thrombosis via its interaction with the receptors glycoprotein (GP)VI and integrin α₂β₁. Adhesion and degranulation‐promoting adapter protein (ADAP) regulates α_IIbβ₃ in platelets and α_Lβ₂ in T cells, and is phosphorylated in GPVI‐deficient platelets activated by collagen. Objectives: To determine whether ADAP plays a role in collagen‐induced platelet activation and in the regulation and function of α₂β₁. Methods: Using ADAP^?/? mice and synthetic collagen peptides, we investigated the role of ADAP in platelet aggregation, adhesion, spreading, thromboxane synthesis, and tyrosine phosphorylation. Results and Conclusions: Platelet aggregation and phosphorylation of phospholipase Cγ2 induced by collagen were attenuated in ADAP^?/? platelets. However, aggregation and signaling induced by collagen‐related peptide (CRP), a GPVI‐selective agonist, were largely unaffected. Platelet adhesion to CRP was also unaffected by ADAP deficiency. Adhesion to the α₂β₁‐selective ligand GFOGER and to a peptide (III‐04), which supports adhesion that is dependent on both GPVI and α₂β₁, was reduced in ADAP^?/? platelets. An impedance‐based label‐free detection technique, which measures adhesion and spreading of platelets, indicated that, in the absence of ADAP, spreading on GFOGER was also reduced. This was confirmed with non‐fluorescent differential‐interference contrast microscopy, which revealed reduced filpodia formation in ADAP^?/? platelets adherent to GFOGER. This indicates that ADAP plays a role in mediating platelet activation via the collagen‐binding integrin α₂β₁. In addition, we found that ADAP^?/? mice, which are mildly thrombocytopenic, have enlarged spleens as compared with wild‐type animals. This may reflect increased removal of platelets from the circulation.     

RGD‐ligand mimetic antagonists of integrin αIIbβ3 paradoxically enhance GPVI‐induced human platelet activation

Summary. Background: The integrin α_IIbβ₃ is the major mediator of platelet aggregation and has, therefore, become an important target of antithrombotic therapy. Antagonists of α_IIbβ₃, for example abciximab, tirofiban and eptifibatide, are used in the treatment of acute coronary syndromes. However, in addition to effective blockade of the integrin, binding of can induce conformational changes in the integrin and can also induce integrin clustering. This class effect of RGD‐ligand mimetics might, therefore, underlie paradoxical platelet activation and thrombosis previously reported. Objectives: To examine the components of signaling pathways and functional responses in platelets that may underlie this phenomenon of paradoxical platelet activation. Methods: We assessed the effect of lotrafiban, and other α_IIbβ₃ antagonists including the clinically used drug tirofiban, on tyrosine phosphorylation of key signaling proteins in platelets by immunoblotting and also platelet functional outputs such as cytosolic calcium responses, phosphatidylserine exposure (pro‐coagulant activity) and dense granule release. Results: In all cases, no effect of α_IIbβ₃ antagonists were observed on their own, but these integrin antagonists did lead to a marked potentiation of glycoprotein VI (GPVI)‐associated FcR γ‐chain phosphorylation, activation of Src family kinases and Syk kinase. This correlated with increased dense granule secretion, cytosolic calcium response and exposure of phosphatidylserine on the platelet surface. P2Y₁₂ antagonism abolished the potentiated phosphatidylserine exposure and dense granule secretion but not the cytosolic calcium response. Conclusions: These data provide a mechanism for enhancement of platelet activity by α_IIbβ₃ inhibitors, but also reveal a potentially important signaling pathway operating from the integrin to GPVI signaling.     

CD40 ligand shedding is regulated by interaction between matrix metalloproteinase‐2 and platelet integrin αIIbβ3

Summary. Background: CD40 ligand (CD40L, CD154) in the circulatory system is mainly contained in platelets, and surface‐expressed CD40L on activated platelets is subsequently cleaved by proteolytic activity to generate soluble CD40L (sCD40L). However, the enzyme responsible for the shedding of CD40L in activated platelets has not been clearly identified yet. We have recently found that molecular interaction of matrix metalloproteinase‐2 (MMP‐2) with integrin α_IIbβ₃ is required for the enhancement of platelet activation. Objectives: To elucidate the biochemical mechanism of MMP‐2‐associated sCD40L release. Methods: Localization of MMP‐2 and CD40L in platelets was analyzed by flow cytometry and fluorescence microscopy. The release of sCD40L from activated platelets was measured by enzyme‐linked immunosorbent assay. MMP‐2 binding to α_IIbβ₃ was analyzed by immunoprecipitation and western blotting. Recombinant hemopexin‐like domain and MMP‐2‐specific inhibitor were used to characterize the nature of MMP‐2 binding and catalytic activity. Results: It was revealed that interaction of MMP‐2 with α_IIbβ₃ is required for effective production of sCD40L in activated human platelets. Platelet activation and release of sCD40L were significantly affected by inhibition of platelet‐derived MMP‐2 activity or by inhibition of binding between the enzyme and the integrin. It was also found in platelet‐rich plasma that MMP‐2 activity is responsible for generating sCD40L. Conclusions: The results presented here strongly suggest that MMP‐2 interacts with α_IIbβ₃ to regulate the shedding of CD40L exposed on the surfaces of activated human platelets.     

PECAM‐1 functions as a negative regulator of laminin‐induced platelet activation

Summary. Background: Interaction of resting platelets with exposed components of the subendothelial matrix is an important early activating event that takes place at sites of vascular injury. Platelet responses to collagen are mediated by integrin α₂β₁ and the glycoprotein (GP)VI–Fc receptor (FcR) γ‐chain complex, whereas platelet activation by laminin is mediated by the related integrin, α₆β₁, and similarly requires signaling through GPVI–FcR γ‐chain. Objective: Because the cell adhesion and signaling receptor PECAM‐1 has previously been shown to dampen collagen‐induced platelet activation, we sought to determine whether PECAM‐1 might similarly regulate platelet activation by laminin. Methods/Results: We found that PECAM‐1 became tyrosine phosphorylated on its cytoplasmic immunoreceptor tyrosine‐based inhibitory motifs following adhesion of either human or murine platelets to immobilized laminin. Whereas the presence or absence of PECAM‐1 had no effect on either the rate or extent of platelet adhesion or spreading on laminin, PECAM‐1 inhibited laminin‐induced phosphorylation of GPVI–FcR γ‐chain immunoreceptor tyrosine‐based activation motifs (ITAMs) and activation of its downstream effector, Syk kinase, and suppressed granule secretion. Conclusions: Taken together, these data are consistent with previous findings in platelets and other blood and vascular cells that PECAM‐1 functions by modulating ITAM‐mediated signaling pathways that amplify cellular activation.     

Succinate independently stimulates full platelet activation via cAMP and phosphoinositide 3‐kinase‐β signaling

Summary. Background: The citric cycle intermediate succinate has recently been identified as a ligand for the G‐protein‐coupled receptor (GPCR) SUCNR1. We have previously found that this receptor is one of the most highly expressed GPCRs in human platelets. Objective: The aim of this study was to investigate the role of SUCNR1 in platelet aggregation and to explore the signaling pathways of this receptor in platelets. Methods and Results: Using real‐time‐PCR, we demonstrated that SUCNR1 is expressed in human platelets at a level corresponding to that of the P2Y₁ receptor. Light transmission aggregation experiments showed dose‐dependent aggregation induced by succinate, reaching a maximum response at 0.5 mm . The effect of succinate on platelet aggregation was confirmed with flow cytometry, showing increased surface expression of activated glycoprotein IIb–IIIa and P‐selectin. Intracellular SUCNR1 signaling was found to result in decreased cAMP levels, Akt phosphorylation mediated by phosphoinositide 3‐kinase‐β activation, and receptor desensitization. Furthermore, succinate‐induced platelet aggregation was demonstrated to depend on Src, generation of thromboxane A₂, and ATP release. Platelet SUCNR1 is subject to desensitization through both homologous and heterologous mechanisms. In addition, the P2Y₁₂ receptor inhibitor ticagrelor completely prevented platelet aggregation induced by succinate. Conclusions: Our experiments show that succinate induces full aggregation of human platelets via SUCNR1. Succinate‐induced platelet aggregation depends on thromboxane A₂ generation, ATP release, and P2Y₁₂ activation.     

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.

     

Integrin α2β1 induces phosphorylation-dependent and phosphorylation-independent activation of phospholipase Cγ2 in platelets: role of Src kinase and Rac GTPase

Summary. Background: Platelet adhesion promoted by integrin  α₂β₁ induces integrin  α_IIbβ₃ activation through the phospholipase C (PLC)‐dependent stimulation of the small GTPase Rap1b. Objective: To analyze the mechanism of PLC activation downstream of α₂β₁ that is required for regulation of Rap1b and α_IIbβ₃. Methods: Human and murine platelets were allowed to adhere to immobilized type I monomeric collagen through α₂β₁. Tyrosine phosphorylation of PLCγ2, PLC activation, accumulation of GTP‐bound Rap1b and fibrinogen binding were measured and compared. Results: Integrin  α₂β₁ recruitment induced an evident PLC activation that was concomitant with robust tyrosine phosphorylation of PLCγ2, and was suppressed in platelets from PLCγ2‐knockout mice. Moreover, PLCγ2^?/? platelets were unable to accumulate active Rap1b and to activate α_IIbβ₃ upon adhesion through α₂β₁. Inhibition of Src kinases completely prevented tyrosine phosphorylation of PLCγ2 in adherent platelets, but did not affect its activation, and both Rap1b and α_IIbβ₃ stimulation occurred normally. Importantly, α_IIbβ₃‐induced phosphorylation and activation of PLCγ2, as well as accumulation of active Rap1b, were totally suppressed by Src inhibition. Integrin  α₂β₁ recruitment triggered the Src kinase‐independent activation of the small GTPase Rac1, and activation of Rac1 was not required for PLCγ2 phosphorylation. However, when phosphorylation of PLCγ2 was blocked by the Src kinase inhibitor PP2, prevention of Rac1 activation significantly reduced PLCγ2 activation, GTP‐Rap1b accumulation, and α_IIbβ₃ stimulation. Conclusions: Src kinases and the Rac GTPases mediate independent pathways for PLCγ2 activation downstream of α₂β₁.     

Relative contributions of stromal interaction molecule 1 and CalDAG‐GEFI to calcium‐dependent platelet activation and thrombosis

Summary. Background: Stromal interaction molecule 1 (STIM1) was recently identified as a critical component of store‐operated calcium entry (SOCE) in platelets. We previously reported the Ca²⁺‐sensing guanine nucleotide exchange factor CalDAG‐GEFI as a critical molecule in Ca²⁺ signaling in platelets. Objective: To evaluate the contribution of STIM1/SOCE to Ca²⁺‐dependent platelet activation and thrombosis, we here compared the activation responses of platelets lacking STIM1 and platelets lacking CalDAG‐GEFI. Methods: The murine Stim1 gene was conditionally deleted in the megakaryocyte/platelet lineage. CalDAG‐GEFI^–/– and Stim1^fl/flPF4‐Cre mice, along with littermate control mice, were used for in vitro and in vivo experiments under flow as well as static conditions. Results: Integrin α_IIbβ₃‐mediated aggregation was markedly impaired in CalDAG‐GEFI‐deficient but not STIM1‐deficient platelets, under both static and flow conditions. In contrast, deficiency in either STIM1 or CalDAG‐GEFI significantly impaired the ability of platelets to express phosphatidylserine on the cell surface. When subjected to a laser injury thrombosis model, mice lacking STIM1 in platelets were characterized by the formation of unstable platelet‐rich thrombi and delayed and reduced fibrin generation in injured arterioles. In CalDAG‐GEFI^–/– mice, fibrin generation was also delayed and reduced, but platelet accumulation was almost abolished. Conclusions: Our studies suggest that: (i) STIM1/SOCE is critical for the procoagulant activity but not the proadhesive function of platelets; and (ii) at the site of vascular injury, STIM1 and CalDAG‐GEFI are critical for the first wave of thrombin generation mediated by procoagulant platelets.     

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.     

ITAM receptor‐mediated generation of reactive oxygen species in human platelets occurs via Syk‐dependent and Syk‐independent pathways

Background: Ligation of the platelet‐specific collagen receptor, GPVI/FcRγ, causes rapid, transient disulfide‐dependent homodimerization, and the production of intracellular reactive oxygen species (ROS) generated by the NADPH oxidase, linked to GPVI via TRAF4. Objectives: The aim of this study was to evaluate the role of early signaling events in ROS generation following engagement of either GPVI/FcRγ or a second immunoreceptor tyrosine‐based activation motif (ITAM)‐containing receptor on platelets, FcγRIIa. Methods and Results: Using an H₂DCF‐DA‐based flow cytometric assay to measure intracellular ROS, we show that treatment of platelets with either the GPVI agonists, collagen‐related peptide (CRP) or convulxin (Cvx), or the FcγRIIa agonist 14A2, increased intraplatelet ROS; other platelet agonists such as ADP and TRAP did not. Basal ROS in platelet‐rich plasma from 14 healthy donors displayed little inter‐individual variability. CRP, Cvx or 14A2 induced an initial burst of ROS within 2 min followed by additional ROS reaching a plateau after 15–20 min. The Syk inhibitor BAY61‐3606, which blocks ITAM‐dependent signaling, had no effect on the initial ROS burst, but completely inhibited the second phase. Conclusions: Together, these results show for the first time that ROS generation downstream of GPVI or FcγRIIa consists of two distinct phases: an initial Syk‐independent burst followed by additional Syk‐dependent generation.     

Prohibitins are involved in protease‐activated receptor 1‐mediated platelet aggregation

Summary. Background: Prohibitins (PHBs), comprising the two homologous members PHB1 and PHB2, are ubiquitously expressed and highly conserved. The membrane PHBs have been reported to be involved in typhoid fever, obesity, and cancer metastasis. Proteomic studies have revealed the presence of PHBs in human platelets, but the roles of PHBs during platelet aggregation are unknown.Objectives: To investigate the role of PHBs in platelet aggregation. Methods and results: PHB1 and PHB2 were detected on the surfaces of human platelets by flow cytometry and confocal microscopy. The PHBs were distributed in lipid rafts, as determined by sucrose density centrifugation. In addition, the PHBs were associated with protease‐activated receptor 1 (PAR1), as determined by Bm‐TFF2 (a PAR1 agonist)‐affinity chromatography, coimmunoprecipitation, and confocal microscopy. The platelet aggregation, α_IIbβ₃ activation, granular secretion and calcium mobilization stimulated by low concentrations of thrombin (0.05 U mL^?1) or PAR1‐activating peptide (PAR1‐AP) (20 μm ) were reduced or abolished as a result of the blockade of PHBs by anti‐PHB antibodies or their Fab fragments; however, the same results were not obtained with induction by high concentrations of thrombin (0.6 U mL^?1) or protease‐activated receptor 4‐activating peptide (300 μm ). The calcium mobilization in MEG‐01 megakaryocytes stimulated by PAR1‐AP was significantly suppressed by PHB depletion with RNA interference against PHB1 and PHB2. Conclusions: PHBs are localized on the human platelet membrane and are involved in PAR1‐mediated platelet aggregation. Until recently, PHBs were unknown as regulators of PAR1 signaling, and they may be effective targets for antiplatelet therapy.     

Nitric oxide inhibits von Willebrand factor‐mediated platelet adhesion and spreading through regulation of integrin αIIbβ3 and myosin light chain

Summary. Background: von Willebrand factor (VWF)‐mediated platelet adhesion and spreading at sites of vascular injury is a critical step in hemostasis. This process requires two individual receptors: glycoprotein Ib (GPIb)‐V‐IX and integrin α_IIbβ₃. However, little is known about the negative regulation of these events. Objectives: To examine if the endogenous platelet inhibitor nitric oxide (NO) has differential effects on adhesion, spreading and aggregation induced by immobilized VWF. Results: S‐nitrosoglutathione (GSNO) inhibited platelet aggregation on immobilized VWF under static and flow conditions, but had no effect on platelet adhesion. Primary signaling events underpinning the actions of NO required cyclic GMP but not protein kinase A. Dissecting the roles of GPIb and integrin α_IIbβ₃ demonstrated that NO targeted α_IIbβ₃‐mediated aggregation and spreading, but did not significantly influence GPIb‐mediated adhesion. To understand the relationship between the effects of NO on adhesion and subsequent aggregation, we evaluated the activation of α_IIbβ₃ on adherent platelets. NO reduced the phosphorylation of extracellular stimuli‐responsive kinase (ERK) and p38, required for integrin activation resulting in reduced binding of the activated α_IIbβ₃‐specific antibody PAC‐1 on adherent platelets. Detailed analysis of platelet spreading initiated by VWF demonstrated key roles for integrin α_IIbβ₃ and myosin light chain (MLC) phosphorylation. NO targeted both of these pathways by directly modulating integrin affinity and activating MLC phosphatase. Conclusion: These data demonstrate that initial activation‐independent platelet adhesion to VWF via GPIb is resistant to NO, however, NO inhibits GPIb‐mediated activation of α_IIbβ₃ and MLC leading to reduced platelet spreading and aggregation.     

A Gi‐independent mechanism mediating Akt phosphorylation in platelets

Summary. Background: The serine‐threonine kinase Akt plays an important role in regulating platelet activation. Stimulation of platelets with various agonists results in Akt activation as indicated by Akt phosphorylation. However, the mechanisms of Akt phosphorylation in platelets are not completely understood. Objectives and Methods: We used P2Y₁₂ knockout mice to address the role of P2Y₁₂ in Akt phosphorylation in response to thrombin receptors in platelets. Results: Thrombin or the PAR4 thrombin receptor peptide AYPGKF at high concentrations stimulated substantial phosphorylation of Akt residues Thr³⁰⁸ and Ser⁴⁷³ in P2Y₁₂‐deficient platelets. AYPGKF‐induced Akt phosphorylation is enhanced by expression of recombinant human PAR4 cDNA in Chinese hamster ovary (CHO) cells. P2Y₁₂‐independent Akt phosphorylation was not inhibited by integrin inhibitor peptide RGDS or integrin β₃ deficiency. Akt phosphorylation induced by thrombin or AYPGKF in P2Y₁₂‐deficient platelets was inhibited by the calcium chelator dimethyl‐BAPTA, the Src family kinase inhibitor PP2, and PI3K inhibitors, respectively. Conclusions: Our results reveal a novel P2Y₁₂‐independent signaling pathway mediating Akt phosphorylation in response to thrombin receptors.     

Diverse functions of protein kinase C isoforms in platelet activation and thrombus formation

Summary. Platelet activation is a complex balance of positive and negative signaling pathways. The protein kinase C (PKC) family is a major regulator of platelet granule secretion, integrin activation, aggregation, spreading and procoagulant activity. As broad‐spectrum PKC inhibitors reduce secretion and aggregation, the PKC family is generally considered to be a positive regulator of platelet activation. However, the individual members of the PKC family that are expressed in platelets are regulated in different ways, and an increasing body of evidence indicates that they have distinct, and often opposing, roles. Many of the recent advances in understanding the contributions of individual PKC isoforms have come from mouse gene knockout studies. PKCα, a classic isoform, is an essential positive regulator of granule secretion and thrombus formation, both in vitro and in vivo. Mice lacking PKCα show much reduced thrombus formation in vivo but do not have a bleeding defect, suggesting that PKCα could be an attractive antithrombotic target. Important, apparently non‐redundant, roles, both positive and negative, for the novel PKC isoforms δ, θ and ε in granule secretion have also been proposed, indicating highly complex regulation of this essential process. Similarly, PKCβ, PKCδ and PKCθ have non‐redundant roles in platelet spreading, as absence of either PKCβ or PKCθ reduces spreading, whereas PKCδ negatively regulates filopodial formation. This negative signaling by PKCδ may reduce platelet aggregation and so restrict thrombus formation. In this review, we discuss the current understanding of the regulation and functions of individual PKC isoforms in platelet activation and thrombus formation.     

Novel aspects of Kindlin‐3 function in humans based on a new case of leukocyte adhesion deficiency III

Summary. Background: Kindlin‐3 is a novel integrin activator in hematopoietic cells, and its deficiency leads to immune problems and severe bleeding, known as leukocyte adhesion deficiency III (LAD‐III). Our current understanding of Kindlin‐3 function primarily relies on analysis of animal models or cell lines. Objectives: To understand the functions of Kindlin‐3 in human primary blood cells. Patients/Methods: We analyzed primary and immortalized hematopoietic cells obtained from a new LAD‐III patient with immune problems, bleeding, a history of anemia, and abnormally shaped red blood cells. Results: The patient’s white blood cells (WBCs) and platelets showed defects in agonist‐induced integrin activation and botrocetin‐induced platelet agglutination. Primary leukocytes from this patient exhibited abnormal activation of β₁ integrin. Integrin activation defects were responsible for the observed deficiency in the botrocetin‐induced platelet response. Analysis of patient genomic DNA revealed a novel mutation in the Kindlin3 gene. The mutation abolished Kindlin‐3 expression in primary WBCs and platelets, owing to abnormal splicing. Kindlin‐3 is expressed in red blood cells (RBCs), and its deficiency is proposed to lead to abnormally shaped RBCs. Immortalized patient WBCs expressed a truncated form of Kindlin‐3 that was not sufficient to support integrin activation. Expression of Kindlin‐3 cDNA in immortalized patient WBCs rescued integrin activation defects, whereas overexpression of the truncated form did not. Conclusions: Kindlin‐3 deficiency impairs integrin function, including activation of β₁ integrin. Abnormalities in glycoprotein Ib–IX function in Kindlin‐3‐deficient platelets are secondary to integrin defects. The region of Kindlin‐3 encoded by exon 11 is crucial for its ability to activate integrins in humans.     

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.     

PKC inhibition markedly enhances Ca2+ signaling and phosphatidylserine exposure downstream of protease‐activated receptor‐1 but not protease‐activated receptor‐4 in human platelets

To cite this article: Harper MT, Poole AW. PKC inhibition markedly enhances Ca²⁺ signaling and phosphatidylserine exposure downstream of protease‐activated receptor‐1 but not protease‐activated receptor‐4 in human platelets. J Thromb Haemost 2011; 9 : 1599–607. Summary. Background: Cytosolic calcium concentration is a critical regulator of platelet activation, and so platelet Ca²⁺ signaling must be tightly controlled. Thrombin‐induced Ca²⁺ signaling is enhanced by inhibitors of protein kinase C (PKC), suggesting that PKC negatively regulates the Ca²⁺signal, although the mechanisms by which this occurs and its physiological relevance are still unclear. Objectives: To investigate the mechanisms by which PKC inhibitors enhance thrombin‐induced Ca²⁺ signaling, and to determine the importance of this pathway in platelet activation. Methods: Cytosolic Ca²⁺ signaling was monitored in fura‐2‐loaded human platelets. Phosphatidylserine (PS) exposure, a marker of platelet procoagulant activity, was measured by annexin V binding and flow cytometry. Results: PKC inhibition by bisindolylmaleimide‐I (BIM‐I) enhanced α‐thrombin‐induced Ca²⁺ signaling in a concentration‐dependent manner. PAR1 signaling, activated by SFLLRN, was enhanced much more strongly than PAR4, activated by AYPGKF or γ‐thrombin, which is a potent PAR4 agonist but a poor activator of PAR1. BIM‐I had little effect on α‐thrombin‐induced signaling following treatment with the PAR1 antagonist, SCH‐79797. BIM‐I enhanced Ca²⁺ release from intracellular stores and Ca²⁺ entry, as assessed by Mn²⁺ quench. However, the plasma membrane Ca²⁺ ATPase inhibitor, 5(6)‐carboxyeosin, did not prevent the effect of BIM‐I. PKC inhibition strongly enhanced α‐thrombin‐induced PS exposure, which was reversed by blockade of PAR1. Conclusions: Together, these data show that when PAR1 is stimulated, PKC negatively regulates Ca²⁺ release and Ca²⁺ entry, which leads to reduced platelet PS exposure.