Matthew T. Harper

PKCα regulates platelet granule secretion and thrombus formation in mice

Platelets are central players in atherothrombosis development in coronary artery disease. The PKC family provides important intracellular mechanisms for regulating platelet activity, and platelets express several members of this family, including the classical isoforms PKCalpha and PKCbeta and novel isoforms PKCdelta and PKCtheta. Here, we used a genetic approach to definitively demonstrate the role played by PKCalpha in regulating thrombus formation and platelet function. Thrombus formation in vivo was attenuated in Prkca-/- mice, and PKCalpha was required for thrombus formation in vitro, although this PKC isoform did not regulate platelet adhesion to collagen. The ablation of in vitro thrombus formation in Prkca-/- platelets was rescued by the addition of ADP, consistent with the key mechanistic finding that dense-granule biogenesis and secretion depend upon PKCalpha expression. Furthermore, defective platelet aggregation in response to either collagen-related peptide or thrombin could be overcome by an increase in agonist concentration. Evidence of overt bleeding, including gastrointestinal and tail bleeding, was not seen in Prkca-/- mice. In summary, the effects of PKCalpha ablation on thrombus formation and granule secretion may implicate PKCalpha as a drug target for antithrombotic therapy.

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.

Functional divergence of platelet protein kinase C (PKC) isoforms in thrombus formation on collagen

Arterial thrombosis, a major cause of myocardial infarction and stroke, is initiated by activation of blood platelets by subendothelial collagen. The protein kinase C (PKC) family centrally regulates platelet activation, and it is becoming clear that the individual PKC isoforms play distinct roles, some of which oppose each other. Here, for the first time, we address all four of the major platelet-expressed PKC isoforms, determining their comparative roles in regulating platelet adhesion to collagen and their subsequent activation under physiological flow conditions. Using mouse gene knock-out and pharmacological approaches in human platelets, we show that collagen-dependent α-granule secretion and thrombus formation are mediated by the conventional PKC isoforms, PKCα and PKCβ, whereas the novel isoform, PKCθ, negatively regulates these events. PKCδ also negatively regulates thrombus formation but not α-granule secretion. In addition, we demonstrate for the first time that individual PKC isoforms differentially regulate platelet calcium signaling and exposure of phosphatidylserine under flow. Although platelet deficient in PKCα or PKCβ showed reduced calcium signaling and phosphatidylserine exposure, these responses were enhanced in the absence of PKCθ. In summary therefore, this direct comparison between individual subtypes of PKC, by standardized methodology under flow conditions, reveals that the four major PKCs expressed in platelets play distinct non-redundant roles, where conventional PKCs promote and novel PKCs inhibit thrombus formation on collagen.

Transient Receptor Potential Channels Function as a Coincidence Signal Detector Mediating Phosphatidylserine Exposure

Platelets use the ion channels TRPC3 and TRPC6 to detect simultaneous exposure to two prothrombotic signals. Clotting Takes Two Signals Blood platelets can aggregate to form thrombi to stop blood flow, a necessary physiological process that prevents excessive blood loss but one that can also contribute to blood vessel occlusion and ischemia. Positive feedback loops and integration of multiple signals are mechanisms for regulating biological processes. Harper et al. found that the nonselective cation channels TRPC3 (transient receptor potential C3) and TRPC6 were necessary to enable mouse platelets to expose phosphatidylserine in response to both thrombin and another prothrombotic signal, collagen-related peptide. Sustained calcium influx, which is necessary for phosphatidylserine exposure and thrombin generation, occurred in platelets activated by both thrombin and collagen-related peptide but not in those activated by either signal alone. Understanding how the signaling pathways trigger thrombosis could guide the development of therapies to control blood clotting. Blood platelet aggregation must be tightly controlled to promote clotting at injury sites but avoid inappropriate occlusion of blood vessels. Thrombin, which cleaves and activates Gq-coupled protease-activated receptors, and collagen-related peptide, which activates the receptor glycoprotein VI, stimulate platelets to aggregate and form thrombi. Coincident activation by these two agonists synergizes, causing the exposure of phosphatidylserine on the cell surface, which is a marker of cell death in many cell types. Phosphatidylserine exposure is also essential to produce additional thrombin on platelet surfaces, which contributes to thrombosis. We found that activation of either thrombin receptors or glycoprotein VI alone produced a calcium signal that was largely dependent only on store-operated Ca2+ entry. In contrast, experiments with platelets from knockout mice showed that the presence of both ligands activated nonselective cation channels of the transient receptor potential C (TRPC) family, TRPC3 and TRPC6. These channels principally allowed entry of Na+, which coupled to reverse-mode Na+/Ca2+ exchange to allow calcium influx and thereby contribute to Ca2+ signaling and phosphatidylserine exposure. Thus, TRPC channels act as coincidence detectors to coordinate responses to multiple signals in cells, thereby indirectly mediating in platelets an increase in intracellular calcium concentrations and exposure of prothrombotic phosphatidylserine.

Store-operated calcium entry and non-capacitative calcium entry have distinct roles in thrombin-induced calcium signalling in human platelets

Phosphatidylserine (PS)-exposing platelets accelerate coagulation at sites of vascular injury. PS exposure requires sustained Ca2+ signalling. Two distinct Ca2+ entry pathways amplify and sustain platelet Ca2+ signalling, but their relative importance in human platelets is not known. Here we examined the relative roles of store-operated Ca2+ entry (SOCE) and non-capacitative Ca2+ entry (NCCE) in thrombin-induced Ca2+ signalling and PS exposure by using two Ca2+ channel blockers. BTP-2 showed marked selectivity for SOCE over NCCE. LOE-908 specifically blocked NCCE under our conditions. Using these agents we found that SOCE is important at low thrombin concentrations whereas NCCE became increasingly important as thrombin concentration was increased. PS exposure was reduced by LOE-908, and only activated at thrombin concentrations that also activate NCCE. In contrast, BTP-2 had no effect on PS exposure. We suggest that SOCE amplifies and sustains Ca2+ signalling in response to low concentrations of thrombin whereas both NCCE and SOCE are important contributors to Ca2+ signalling at higher thrombin concentrations. However, despite being involved in Ca2+ signalling at high thrombin concentrations, SOCE is not important for thrombin-induced PS exposure in human platelets. This suggests that the route of Ca2+ entry is an important regulator of thrombin-induced PS exposure in platelets.

Isoform-specific functions of protein kinase C: the platelet paradigm.

Platelets are central to haemostasis and thrombosis. Many key steps in platelet activation and aggregation are regulated by members of the PKC (protein kinase C) family. Multiple isoforms of PKC are expressed in platelets, and evidence is emerging that different isoforms play distinct roles in the platelet activation process. This may, in part, be regulated by isoform-specific interactions between PKC family members and other intracellular signalling molecules, such as tyrosine kinases, or the actin cytoskeleton regulator, VASP (vasodilator-stimulated phosphoprotein). The contributions of individual PKC isoforms can be addressed directly in platelets from knockout mouse models, which are providing key insights into the physiological function of PKC isoform diversity and can be a valuable complimentary approach to more commonly used pharmacological analyses. Using knockout mouse models, recent reports have demonstrated the importance of PKCbeta and PKCtheta in integrin-dependent platelet spreading, and also a novel role for PKCdelta in regulating filopodial formation, highlighting the utility of such models to investigate the functions of specific PKC isoforms in a physiological process that is significant to our understanding of cardiovascular disease.

Canonical Wnt signaling negatively regulates platelet function

Wnts regulate important intracellular signaling events, and dysregulation of the Wnt pathway has been linked to human disease. Here, we uncover numerous Wnt canonical effectors in human platelets where Wnts, their receptors, and downstream signaling components have not been previously described. We demonstrate that the Wnt3a ligand inhibits platelet adhesion, activation, dense granule secretion, and aggregation. Wnt3a also altered platelet shape change and inhibited the activation of the small GTPase RhoA. In addition, we found the Wnt-β-catenin signaling pathway to be functional in platelets. Finally, disruption of the Wnt Frizzled 6 receptor in the mouse resulted in a hyperactivatory platelet phenotype and a reduced sensitivity to Wnt3a. Taken together our studies reveal a novel functional role for Wnt signaling in regulating anucleate platelet function and may provide a tractable target for future antiplatelet therapy.

Protein kinase C mediates platelet secretion and thrombus formation through protein kinase D2

Platelets are highly specialized blood cells critically involved in hemostasis and thrombosis. Members of the protein kinase C (PKC) family have established roles in regulating platelet function and thrombosis, but the molecular mechanisms are not clearly understood. In particular, the conventional PKC isoform, PKCα, is a major regulator of platelet granule secretion, but the molecular pathway from PKCα to secretion is not defined. Protein kinase D (PKD) is a family of 3 kinases activated by PKC, which may represent a step in the PKC signaling pathway to secretion. In the present study, we show that PKD2 is the sole PKD member regulated downstream of PKC in platelets, and that the conventional, but not novel, PKC isoforms provide the upstream signal. Platelets from a gene knock-in mouse in which 2 key phosphorylation sites in PKD2 have been mutated (Ser707Ala/Ser711Ala) show a significant reduction in agonist-induced dense granule secretion, but not in α-granule secretion. This deficiency in dense granule release was responsible for a reduced platelet aggregation and a marked reduction in thrombus formation. Our results show that in the molecular pathway to secretion, PKD2 is a key component of the PKC-mediated pathway to platelet activation and thrombus formation through its selective regulation of dense granule secretion.

Genetic Analysis of the Role of Protein Kinase Cθ in Platelet Function and Thrombus Formation

Background PKCθ is a novel protein kinase C isozyme, predominately expressed in T cells and platelets. PKCθ−/− T cells exhibit reduced activation and PKCθ−/− mice are resistant to autoimmune disease, making PKCθ an attractive therapeutic target for immune modulation. Collagen is a major agonist for platelets, operating through an immunoreceptor-like signalling pathway from its receptor GPVI. Although it has recently been shown that PKCθ positively regulates outside-in signalling through integrin αIIbβ3 in platelets, the role of PKCθ in GPVI-dependent signalling and functional activation of platelets has not been assessed. Methodology/Principal Findings In the present study we assessed static adhesion, cell spreading, granule secretion, integrin αIIbβ3 activation and platelet aggregation in washed mouse platelets lacking PKCθ. Thrombus formation on a collagen-coated surface was assessed in vitro under flow. PKCθ−/− platelets exhibited reduced static adhesion and filopodia generation on fibrinogen, suggesting that PKCθ positively regulates outside-in signalling, in agreement with a previous report. In contrast, PKCθ−/− platelets also exhibited markedly enhanced GPVI-dependent α-granule secretion, although dense granule secretion was unaffected, suggesting that PKCθ differentially regulates these two granules. Inside-out regulation of αIIbβ3 activation was also enhanced downstream of GPVI stimulation. Although this did not result in increased aggregation, importantly thrombus formation on collagen under high shear (1000 s−1) was enhanced. Conclusions/Significance These data suggest that PKCθ is an important negative regulator of thrombus formation on collagen, potentially mediated by α-granule secretion and αIIbβ3 activation. PKCθ therefore may act to restrict thrombus growth, a finding that has important implications for the development and safe clinical use of PKCθ inhibitors.

SDF-1α is a novel autocrine activator of platelets operating through its receptor CXCR4

Platelets store and secrete the chemokine stromal cell-derived factor (SDF)-1α upon platelet activation, but the ability of platelet-derived SDF-1α to signal in an autocrine/paracrine manner mediating functional platelet responses relevant to thrombosis and haemostasis is unknown. We sought to explore the role of platelet-derived SDF-1α and its receptors, CXCR4 and CXCR7 in facilitating platelet activation and determine the mechanism facilitating SDF-1α-mediated regulation of platelet function. Using human washed platelets, CXCR4 inhibition, but not CXCR7 blockade significantly abrogated collagen-mediated platelet aggregation, dense granule secretion and thromboxane (Tx) A2 production. Time-dependent release of SDF-1α from collagen-activated platelets supports a functional role for SDF-1α in this regard. Using an in vitro whole blood perfusion assay, collagen-induced thrombus formation was substantially reduced with CXCR4 inhibition. In washed platelets, recombinant SDF-1α in the range of 20–100 ng/mL− 1 could significantly enhance platelet aggregation responses to a threshold concentration of collagen. These enhancements were completely dependent on CXCR4, but not CXCR7, which triggered TxA2 production and dense granule secretion. Rises in cAMP were significantly blunted by SDF-1α, which could also enhance collagen-mediated Ca(2 +) mobilisation, both of which were mediated by CXCR4. This potentiating effect of SDF-1α primarily required TxA2 signalling acting upstream of dense granule secretion, whereas blockade of ADP signalling could only partially attenuate SDF-1α-induced platelet activation. Therefore, this study supports a potentially novel autocrine/paracrine role for platelet-derived SDF-1α during thrombosis and haemostasis, through a predominantly TxA2-dependent and ADP-independent pathway.