Platelet C3G: a key player in vesicle exocytosis, spreading and clot retraction.

C3G is a Rap1 GEF that plays a pivotal role in platelet-mediated processes such as angiogenesis, tumor growth, and metastasis by modulating the platelet secretome. Here, we explore the mechanisms through which C3G governs platelet secretion. For this, we utilized animal models featuring either overexpression or deletion of C3G in platelets, as well as PC12 cell clones expressing C3G mutants. We found that C3G specifically regulates α-granule secretion via PKCδ, but it does not affect δ-granules or lysosomes. C3G activated RalA through a GEF-dependent mechanism, facilitating vesicle docking, while interfering with the formation of the trans-SNARE complex, thereby restricting vesicle fusion. Furthermore, C3G promotes the formation of lamellipodia during platelet spreading on specific substrates by enhancing actin polymerization via Src and Rac1-Arp2/3 pathways, but not Rap1. Consequently, C3G deletion in platelets favored kiss-and-run exocytosis. C3G also controlled granule secretion in PC12 cells, including pore formation. Additionally, C3G-deficient platelets exhibited reduced phosphatidylserine exposure, resulting in decreased thrombin generation, which along with defective actin polymerization and spreading, led to impaired clot retraction. In summary, platelet C3G plays a dual role by facilitating platelet spreading and clot retraction through the promotion of outside-in signaling while concurrently downregulating α-granule secretion by restricting granule fusion.

The fate of mitochondria during platelet activation.

Blood platelets undergo several successive motor-driven reorganizations of the cytoskeleton when they are recruited to an injured part of a vessel. These reorganizations take place during the platelet activation phase, the spreading process on the injured vessel or between fibrin fibers of the forming clot, and during clot retraction. All these steps require a lot of energy, especially the retraction of the clot when platelets develop strong forces similar to those of muscle cells. Platelets can produce energy through glycolysis and mitochondrial respiration. However, although resting platelets have only 5 to 8 individual mitochondria, they produce adenosine triphosphate predominantly via oxidative phosphorylation. Activated, spread platelets show an increase in size compared with resting platelets, and the question arises as to where the few mitochondria are located in these larger platelets. Using expansion microscopy, we show that the number of mitochondria per platelet is increased in spread platelets. Live imaging and focused ion beam-scanning electron microscopy suggest that a mitochondrial fission event takes place during platelet activation. Fission is Drp1 dependent because Drp1-deficient platelets have fused mitochondria. In nucleated cells, mitochondrial fission is associated with a shift to a glycolytic phenotype, and using clot retraction assays, we show that platelets have a more glycolytic energy production during clot retraction and that Drp1-deficient platelets show a defect in clot retraction.

Systemic Review of Clot Retraction Modulators.

Through a process termed clot retraction, platelets cause thrombi to shrink and become more stable. After platelets are activated via inside-out signaling, glycoprotein αIIbßIII binds to fibrinogen and initiates a cascade of intracellular signaling that ends in actin remodeling, which causes the platelet to change its shape. Clot retraction is also important for wound healing. Although the detailed molecular biology of clot retraction is only partially understood, various substances and physiological conditions modulate clot retraction. In this review, we describe some of the current literature pertaining to clot retraction modulators. In addition, we discuss compounds from Cudrania trucuspidata, Arctium lappa, and Panax ginseng that diminish clot retraction and have numerous other health benefits. Caffeic acid and diindolylmethane, both common in plants and vegetables, likewise reduce clot retraction, as do all-trans retinoic acid (a vitamin A derivative), two MAP4K inhibitors, and the chemotherapeutic drug Dasatinib. Conversely, the endogenous anticoagulant Protein S (PS) and the matricellular protein secreted modular calcium-binding protein 1 (SMOC1) both enhance clot retraction. Most studies aiming to identify mechanisms of clot retraction modulators have focused on the increased phosphorylation of vasodilator-stimulated phosphoprotein and inositol 1,4,5-triphosphate receptor I and the decreased phosphorylation of various phospholipases (e.g., phospholipase A2 (PLA2) and phosphatidylinositol-specific phospholipase Cγ2 (PLCγ2), c-Jun N-terminal kinase, and (PI3Ks). One study focused on the decreased phosphorylation of Sarcoma Family Kinases (SFK), and others have focused on increased cAMP levels and the downregulation of inflammatory markers such as thromboxanes, including thromboxane A2 (TXA2) and thromboxane B2 (TXB2); prostaglandin A2 (PGE2); reactive oxygen species (ROS); and cyclooxygenase (COX) enzyme activity. Additionally, pregnancy, fibrinolysis, and the autoimmune condition systemic lupus erythematosus all seem to affect, or at least have some relation with, clot retraction. All the clot retraction modulators need in-depth study to explain these effects.

Molecular basis of clot retraction and its role in wound healing.

Clot retraction is important for the prevention of bleeding, in the manifestations of thrombosis and for tissue repair. The molecular mechanisms behind clot formation are complex. Platelet involvement begins with adhesion at sites of vessel injury followed by platelet aggregation, thrombin generation and fibrin production. Other blood cells incorporate into a fibrin mesh that is consolidated by FXIIIa-mediated crosslinking and platelet contractile activity. The latter results in the asymmetric redistribution of erythrocytes into a tighter central mass providing the clot with stability and resistance to fibrinolysis. Integrin αIIbß3 on platelets is the key player in these events, bridging fibrin and the platelet cytoskeleton. Glycoprotein VI participates in thrombus formation but not in the retraction. Rheological and environmental factors influence clot construction with retraction driven by the platelet cytoskeleton with actomyosin acting as the motor. Activated platelets provide procoagulant activity stimulating thrombin generation together with the release of a plethora of biologically active proteins and substances from storage pools; many form chemotactic gradients within the fibrin or the underlying matrix. Also released are newly synthesized metabolites and lipid-rich vesicles that circulate within the vasculature and mimic platelet functions. Platelets and their released elements play key roles in wound healing. This includes promoting stem cell and mesenchymal stromal cell recruitment, fibroblast and endothelial cell migration, angiogenesis and matrix formation. These properties have led to the use of autologous clots in therapies designed to accelerate tissue repair while offering the potential for genetic manipulation in both inherited and acquired diseases.

Quantification and optimization of clot retraction in washed human platelets by Sonoclot coagulation analysis.

INTRODUCTION: Clot retraction is a pivotal process for haemostasis, where platelets develop a contractile force in fibrin meshwork and lead to the increased rigidity of clot. The pathophysiological alteration in contractile forces generated by the platelet-fibrin meshwork can lead to haemostatic disorders. Regardless of its utter significance, clot retraction remains a limited understood process owing to lack of quantification methodology. Sonoclot analysis is a point-of-care technique used in clinical laboratories for whole blood analysis that provides in vitro qualitative as well as quantitative assessment of coagulation process from initial fibrin formation to clot retraction. METHODS: Human washed platelets were isolated by differential centrifugation method and analysed via optical imaging, microscopy and Sonoclot analysis using 1-2 × 108 /mL of washed platelets, 1 U/mL of thrombin, 1 mg/mL of fibrinogen and 1 mM of calcium chloride. RESULTS: In this study, we demonstrate the novelty of this instrument in the quantitative evaluation of clot retraction in washed platelets and attempted to optimize the reference range of Sonoclot parameters including ACT - 87.3 ± 20.997, CR - 16.23 ± 3.538 and PF - 3.57 ± 0.629, (n = 10). DISCUSSION: Sonoclot analysis provides a simple and quantitative method to better understand in vitro clot retraction and its modulation by retraction components including platelet count, fibrinogen and platelet-fibrin interaction compared with existing conventional methods. Sonoclot may prove to be a valuable tool in thrombus biology research to understand fundamental basis of blood clot retraction.

Paxillin binding to the PH domain of kindlin-3 in platelets is required to support integrin αIIbß3 outside-in signaling.

BACKGROUND: Kindlin-3 is essential for supporting the bidirectional signaling of integrin αIIbß3 in platelets by bridging the crosstalk between integrin αIIbß3 and the cytoplasmic signaling adaptors. OBJECTIVE: In this study, we identified a previously unrecognized paxillin binding site in the pleckstrin homology (PH) domain of kindlin-3 and verified its functional significance. METHODS: Structure-based approaches were employed to identify the paxillin binding site in the PH domain of kindlin-3. In addition, the bidirectional signaling of integrin αIIbß3 were evaluated in both human and mouse platelets. RESULTS: In brief, we found that a ß1-ß2 loop in the PH domain of kindlin-3, an important part of the canonical membrane phospholipid binding pocket, was also involved in mediating paxillin interaction. Interestingly, the binding sites of paxillin and membrane phospholipids in the PH domain of kindlin-3 were mutually exclusive. Specific disruption of paxillin binding to the PH domain by point mutations inhibited platelet spreading on immobilized fibrinogen while having no inhibition on soluble fibrinogen binding to stimulated platelets. In addition, a membrane-permeable peptide derived from the ß1-ß2 loop in the PH domain of kindlin-3 was capable of inhibiting platelet spreading and clot retraction, but it had no effect on soluble fibrinogen binding to platelets and platelet aggregation. Treatment with this peptide significantly reduced thrombus formation in mice. CONCLUSION: Taken together, these findings suggest that interaction between paxillin and the PH domain of kindlin-3 plays an important role in supporting integrin αIIbß3 outside-in signaling in platelets, thus providing a novel antithrombotic target.

Asymmetrical Forces Dictate the Distribution and Morphology of Platelets in Blood Clots.

Primary hemostasis consists in the activation of platelets, which spread on the exposed extracellular matrix at the injured vessel surface. Secondary hemostasis, the coagulation cascade, generates a fibrin clot in which activated platelets and other blood cells get trapped. Active platelet-dependent clot retraction reduces the clot volume by extruding the serum. Thus, the clot architecture changes with time of contraction, which may have an important impact on the healing process and the dissolution of the clot, but the precise physiological role of clot retraction is still not completely understood. Since platelets are the only actors to develop force for the retraction of the clot, their distribution within the clot should influence the final clot architecture. We analyzed platelet distributions in intracoronary thrombi and observed that platelets and fibrin co-accumulate in the periphery of retracting clots in vivo. A computational mechanical model suggests that asymmetric forces are responsible for a different contractile behavior of platelets in the periphery versus the clot center, which in turn leads to an uneven distribution of platelets and fibrin fibers within the clot. We developed an in vitro clot retraction assay that reproduces the in vivo observations and follows the prediction of the computational model. Our findings suggest a new active role of platelet contraction in forming a tight fibrin- and platelet-rich boundary layer on the free surface of fibrin clots.

The Antithrombotic Agent Pterostilbene Interferes with Integrin αIIbß3-Mediated Inside-Out and Outside-In Signals in Human Platelets.

Platelets play a crucial role in the physiology of primary hemostasis and pathological processes such as arterial thrombosis; thus, developing a therapeutic target that prevents platelet activation can reduce arterial thrombosis. Pterostilbene (PTE) has remarkable pharmacological activities, including anticancer and neuroprotection. Few studies have reported the effects of pterostilbene on platelet activation. Thus, we examined the inhibitory mechanisms of pterostilbene in human platelets and its role in vascular thrombosis prevention in mice. At low concentrations (2-8 µM), pterostilbene strongly inhibited collagen-induced platelet aggregation. Furthermore, pterostilbene markedly diminished Lyn, Fyn, and Syk phosphorylation and hydroxyl radical formation stimulated by collagen. Moreover, PTE directly hindered integrin αIIbß3 activation through interfering with PAC-1 binding stimulated by collagen. In addition, pterostilbene affected integrin αIIbß3-mediated outside-in signaling, such as integrin ß3, Src, and FAK phosphorylation, and reduced the number of adherent platelets and the single platelet spreading area on immobilized fibrinogen as well as thrombin-stimulated fibrin clot retraction. Furthermore, pterostilbene substantially prolonged the occlusion time of thrombotic platelet plug formation in mice. This study demonstrated that pterostilbene exhibits a strong activity against platelet activation through the inhibition of integrin αIIbß3-mediated inside-out and outside-in signaling, suggesting that pterostilbene can serve as a therapeutic agent for thromboembolic disorders.

A new function for MAP4K4 inhibitors during platelet aggregation and platelet-mediated clot retraction.

Mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) is implicated in type 2 diabetes mellitus, insulin tolerance, inflammation, cancer, and atherosclerosis. We found that GNE 495 and PF 06260933 (both potent and selective MAP4K4 inhibitors) regulated human platelet activation. Immunoblotting revealed human platelets express MAP4K4, and that GNE 495 and PF 06260933 inhibited collagen-, ADP-, and thrombin-induced platelet aggregation and eventually suppressed granule release, TXA2 generation, integrin αIIbß3 activation, and clot retraction. In addition, both inhibitors elevated intracellular levels of cAMP, and coincubation with GNE 495 and aspirin or dipyridamole (a phosphodiesterase inhibitor) synergistically inhibited collagen-induced platelet aggregation and TXA2 generation. Moreover, both inhibitors phosphorylated VASP (ser157), IP3 receptor, and PKA and attenuated MAPK and PI3K/Akt/GSK3ß signaling pathways. This study is the first to demonstrate that MAP4K4 inhibitors reduce thrombus formation by inhibiting platelet activation. These findings also suggest MAP4K4 be considered an emerging target protein for the treatment of thrombosis.

Characterisation of antithrombin-dependent anticoagulants through clot waveform analysis to potentially distinguish them from antithrombin-independent inhibitors targeting activated coagulation factors.

AIMS: While antithrombin (AT)-independent inhibitors targeting thrombin or activated factor X have been assessed through clot waveform (CWA), there are no reports on assessment with respect to AT-dependent anticoagulants. The present study aims to characterise AT-dependent anticoagulants through CWA to distinguish them from AT-independent inhibitors. METHODS: CWA was applied to the activated partial thromboplastin time (APTT) assay of plasma samples spiked with each of AT-dependent drugs (unfractionated heparin, enoxaparin and fondaparinux) and AT-independent drugs (rivaroxaban, apixaban, edoxaban, dabigatran, argatroban, hirudin and bivalirudin), which was performed using the CS-5100 or CN-6000 (Sysmex). The APTT-CWA data were automatically gained by the analyser program. The positive mode of clotting reaction curves was defined as the direction towards fibrin generation. RESULTS: Regarding dose-response curves in AT-dependent anticoagulants, the maximum positive values of the first and secondary derivatives (Max1 and Maxp2, respectively) and the maximum negative values of the secondary derivative (Maxn2) seemed to drop to zero without making an asymptotic line, consistent with the irreversibility. Such a feature was observed also in hirudin, as reported previously. Notably, the symmetric property of Max1 peaks in the waveforms was distorted dose dependently in AT independent but not AT-dependent drugs. A plot of Maxp2 logarithm versus Maxn2 logarithm was linear. The slope was about 1 in AT-dependent drugs while that was more than 1 in AT-independent drugs. These features made it possible to distinguish AT-dependent and AT-independent drugs. CONCLUSIONS: The results aid in further understanding of the pharmacological aspects of anticoagulation and in screening of candidates for novel anticoagulants.

Ultrasound enhanced synthetic platelet therapy for augmented wound repair.

Native platelets perform a number of functions within the wound healing process, including interacting with fibrin fibers at the wound site to bring about retraction after clot formation. Clot retraction improves clot stability and enhances the function of the fibrin network as a provisional matrix to support cellular infiltration of the wound site, thus facilitating tissue repair and remodeling after hemostasis. In cases of traumatic injury or disease, platelets can become depleted and this process disrupted. To that end, our lab has developed synthetic platelet-like particles (PLPs) that recapitulate the clot retraction abilities of native platelets through a Brownian-wrench driven mechanism that drives fibrin network densification and clot retraction over time, however, this Brownian-motion driven process occurs on a longer time scale than native active actin/myosin-driven platelet-mediated clot retraction. We hypothesized that a combinatorial therapy comprised of ultrasound stimulation of PLP motion within fibrin clots would facilitate a faster induction of clot retraction on a more platelet-mimetic time scale and at a lower dosage than required for PLPs acting alone. We found that application of ultrasound in combination with a subtherapeutic dosage of PLPs resulted in increased clot density and stiffness, improved fibroblast migration in vitro and increased epidermal thickness and angiogenesis in vivo, indicating that this combination therapy has potential to facilitate multiphase pro-healing outcomes. Additionally, while these particular studies focus on the role of ultrasound in enhancing specific interactions between fibrin-binding synthetic PLPs embedded within fibrin networks, these studies have wide applicability in understanding the role of ultrasound stimulation in enhancing multi-scale colloidal interactions within fibrillar matrices.

Potential Role of Platelet-Activating C-Type Lectin-Like Proteins in Viper Envenomation Induced Thrombotic Microangiopathy Symptom.

Envenomation by viperid snakes may lead to severe bleeding, consumption coagulopathy, and thrombotic microangiopathy symptoms. The exact etiology or toxins responsible for thrombotic microangiopathy symptoms after snake envenomation remain obscure. Snake C-type lectin-like proteins (snaclecs) are one of the main non-enzymatic protein constituents in viper venoms, of which a majority are considered as modulators of thrombosis and hemostasis. In this study, we demonstrated that two snaclecs (mucetin and stejnulxin), isolated and identified from Protobothrops mucrosquamatus and Trimeresurus stejnegeri venoms, directly induced platelet degranulation and clot-retraction in vitro, and microvascular thrombosis has been confirmed in various organs in vivo. These snaclecs reduced cerebral blood flow and impaired motor balance and spatial memories in mice, which partially represent the thrombotic microangiopathy symptoms in some snakebite patients. The functional blocking of these snaclecs with antibodies alleviated the viper venom induced platelet activation and thrombotic microangiopathy-like symptoms. Understanding the pathophysiology of thrombotic microangiopathy associated with snake envenoming may lead to emerging therapeutic strategies.

Microengineered Flexural Post Rings for Effective Blood Sample Fencing and High-Throughput Measurement of Clot Retraction Force.

During blood clotting, clot retraction alters its mechanical properties and critically affects hemostasis. Despite that, existing clot retraction assays hold limitations such as large footprint and low throughput. In this work, we report the design of flexural post rings for a miniaturized assay of clot retraction force (CRF) with high throughput. Leveraging surface tensions, the post rings hold blood samples in a highly reproducible fashion while simultaneously serving as cantilever beams to measure the CRF. We investigated the effect on the device performance of major parameters, namely, surface hydrophobicity, post number, and post stiffness. We then tested the devices using 14 patient samples and revealed the correlation between CRF and fibrinogen levels. We further implemented an automated liquid handler and developed a high-throughput platform for clot retraction assay. The device's small sample consumption, simple operation, and good compatibility with existing automation facilities make it a promising high-throughput clot retraction assay.

Thromboelastographic Assessment of Fibrinolytic Activity in Postpartum Hemorrhage: A Retrospective Single-Center Observational Study.

BACKGROUND: Postpartum hemorrhage is a leading cause of maternal mortality. Antifibrinolytic therapy has the potential to influence outcomes in postpartum hemorrhage, but the incidence of elevated fibrinolytic activity in postpartum hemorrhage is unknown. METHODS: We retrospectively reviewed thromboelastography (TEG) results obtained for postpartum hemorrhage from 118 deliveries at The University of Chicago. TEG results were obtained as part of our postpartum hemorrhage protocol when blood loss exceeded 500 mL after vaginal delivery or 1000 mL after cesarean delivery. Our primary outcome was the incidence of elevated fibrinolytic activity, which we predefined as clot lysis ≥3% at 30 minutes (Ly30) on kaolin TEG. Platelet-mediated clot retraction can also lead to an elevated Ly30 on kaolin TEG. Therefore, to distinguish between fibrinolysis and clot retraction, we evaluated clot lysis using functional fibrinogen TEG, which contains a platelet inhibitor. We considered a kaolin TEG Ly30 ≥3% in conjunction with a nonzero functional fibrinogen TEG Ly30 suggestive of elevated fibrinolytic activity. We also recorded quantitative blood loss, primary etiology of hemorrhage, standard laboratory measurements of coagulation, and demographic and obstetric characteristics of the study population. RESULTS: The median kaolin TEG Ly30 was 0.2% (interquartile range: 0%-0.8%). Fifteen of 118 women (12.7%; 95% confidence interval, 7.9%-19.9%) had kaolin TEG Ly30 values ≥3%. Of 15 patients with elevated Ly30 values, functional fibrinogen TEG Ly30 was available for 13, of which none demonstrated detectable clot lysis. CONCLUSIONS: Our observation that none of the patients in our sample with kaolin TEG Ly30 values ≥3% had a nonzero functional fibrinogen TEG Ly30 value suggests that the observed elevations in kaolin TEG Ly30 may have been secondary to platelet-mediated clot retraction as opposed to fibrinolysis. Platelet-mediated clot retraction should be distinguished from fibrinolysis when assayed using viscoelastic techniques in postpartum hemorrhage. Further research is necessary to determine the optimal methods to assess fibrinolytic activity in postpartum hemorrhage.

Quantitative Morphology of Cerebral Thrombi Related to Intravital Contraction and Clinical Features of Ischemic Stroke.

BACKGROUND AND PURPOSE: The purpose was to assess quantitatively and qualitatively the composition and structure of cerebral thrombi and correlate them with the signs of intravital clot contraction (retraction), as well as with etiology, severity, duration, and outcomes of acute ischemic stroke. METHODS: We quantified high-resolution scanning electron micrographs of 41 cerebral thrombi for their detailed cellular and noncellular composition and analyzed histological images for the overall structure with the emphasis on red blood cell compression, fibrin age, and the signs of inflammation. RESULTS: Cerebral thrombi were quite compact and had extremely low porosity. The prevailing cell type was polyhedral compressed erythrocytes (polyhedrocytes) in the core, and fibrin-platelet aggregates were concentrated at the periphery; both findings are indicative of intravital contraction of the thrombi. The content of polyhedrocytes directly correlated with the stroke severity. The prevalence of fibrin bundles was typical for more severe cases, while the content of fibrin sponge prevailed in cases with a more favorable course. The overall platelet content in cerebral thrombi was surprisingly small, while the higher content of platelet aggregates was a marker of stroke severity. Fibrillar types of fibrin prevailed in atherothrombogenic thrombi. Older fibrin prevailed in thrombi from the patients who received thrombolytics, and younger fibrin dominated in cardioembolic thrombi. Alternating layers of erythrocytes and fibrin mixed with platelets were common for thrombi from the patients with more favorable outcomes. Thrombi with a higher number of leukocytes were associated with fatal cases. CONCLUSIONS: Most cerebral thrombi undergo intravital clot contraction (retraction) that may be of underestimated clinical importance. Despite the high variability of the composition and structure of cerebral thrombi, the content of certain types of blood cells and fibrin structures combined with the morphological signs of intravital contraction correlate with the clinical course and outcomes of acute ischemic stroke.

The G-protein ßγ subunits regulate platelet function.

AIMS: G-protein coupled receptors (GPCRs) tightly regulate platelet function by interacting with various physiological agonists. An essential mediator of GPCR signaling is the G protein αßγ heterotrimers, in which the ßγ subunits are central players in downstream signaling. Herein, we investigated the role of Gßγ subunits in platelet function, hemostasis and thrombogenesis. METHODS: To achieve this goal, platelets from both mice and humans were employed in the context of a small molecule inhibitor of Gßγ, namely gallein. We used an aggregometer to examine aggregation and dense granules secretion. We also used flow cytometry for P-selectin and PAC1 to determine the impact of inhibiting Gßγ on α -granule secretion and αIIbß3 activation. Clot retraction and the platelet spreading assay were used to examine Gßγ role in outside-in platelet signaling, whereas Western blot was employed to examine its role in Akt activation. Finally, we used the bleeding time assay and the FeCl3-induced carotid-artery injury thrombosis model to determine Gßγ contribution to in vivo platelet function. RESULTS: We observed that gallein inhibits platelet aggregation and secretion in response to agonist stimulation, in both mouse and human platelets. Furthermore, gallein also exerted inhibitory effects on integrin αIIbß3 activation, clot retraction, platelet spreading and Akt activation/phosphorylation. Finally, gallein's inhibitory effects manifested in vivo, as documented by its ability to modulate physiological hemostasis and delay thrombus formation. CONCLUSION: Our findings demonstrate, for the first time, that Gßγ subunits directly regulate GPCR-dependent platelet function, in vitro and in vivo. Moreover, these data highlight Gßγ as a novel therapeutic target for managing thrombotic disorders.

Characterization of Integrin αIIbß3-Mediated Outside-in Signaling by Protein Kinase Cδ in Platelets.

Engagement of integrin αIIbß3 promotes platelet-platelet interaction and stimulates outside-in signaling that amplifies activation. Protein kinase Cδ (PKCδ) is known to play an important role in platelet activation, but its role in outside-in signaling has not been established. In the present study, we determined the role of PKCδ and its signaling pathways in integrin αIIbß3-mediated outside-in signaling in platelets using PKCδ-deficient platelets. Platelet spreading to immobilized fibrinogen resulted in PKCδ phosphorylation, suggesting that αIIbß3 activation caused PKCδ activation. αIIbß3-mediated phosphorylation of Akt was significantly inhibited in PKCδ -/- platelets, indicating a role of PKCδ in outside-in signaling. αIIbß3-mediated PKCδ phosphorylation was inhibited by proline-rich tyrosine kinase 2 (Pyk2) selective inhibitor, suggesting that Pyk2 contributes to the regulation of PKCδ phosphorylation in outside-in signaling. Additionally, Src-family kinase inhibitor PP2 inhibited integrin-mediated Pyk2 and PKCδ phosphorylation. Lastly, platelet spreading was inhibited in PKCδ -/- platelets compared to the wild-type (WT) platelets, and clot retraction from PKCδ -/- platelets was markedly delayed, indicating that PKCδ is involved in the regulation of αIIbß3-dependent interactivities with cytoskeleton elements. Together, these results provide evidence that PKCδ plays an important role in outside-in signaling, which is regulated by Pyk2 in platelets.

Reelin Amplifies Glycoprotein VI Activation and AlphaIIb Beta3 Integrin Outside-In Signaling via PLC Gamma 2 and Rho GTPases.

OBJECTIVE: Reelin, a secreted glycoprotein, was originally identified in the central nervous system, where it plays an important role in brain development and maintenance. In the cardiovascular system, reelin plays a role in atherosclerosis by enhancing vascular inflammation and in arterial thrombosis by promoting platelet adhesion, activation, and thrombus formation via APP (amyloid precursor protein) and GP (glycoprotein) Ib. However, the role of reelin in hemostasis and arterial thrombosis is not fully understood to date. Approach and Results: In the present study, we analyzed the importance of reelin for cytoskeletal reorganization of platelets and thrombus formation in more detail. Platelets release reelin to amplify alphaIIb beta3 integrin outside-in signaling by promoting platelet adhesion, cytoskeletal reorganization, and clot retraction via activation of Rho GTPases RAC1 (Ras-related C3 botulinum toxin substrate) and RhoA (Ras homolog family member A). Reelin interacts with the collagen receptor GP (glycoprotein) VI with subnanomolar affinity, induces tyrosine phosphorylation in a GPVI-dependent manner, and supports platelet binding to collagen and GPVI-dependent RAC1 activation, PLC gamma 2 (1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase gamma-2) phosphorylation, platelet activation, and aggregation. When GPVI was deleted from the platelet surface by antibody treatment in reelin-deficient mice, thrombus formation was completely abolished after injury of the carotid artery while being only reduced in either GPVI-depleted or reelin-deficient mice. CONCLUSIONS: Our study identified a novel signaling pathway that involves reelin-induced GPVI activation and alphaIIb beta3 integrin outside-in signaling in platelets. Loss of both, GPVI and reelin, completely prevents stable arterial thrombus formation in vivo suggesting that inhibiting reelin-platelet-interaction might represent a novel strategy to avoid arterial thrombosis in cardiovascular disease.

The antithrombotic effect of caffeic acid is associated with a cAMP-dependent pathway and clot retraction in human platelets.

Caffeic acid (CA) is a polyphenol widely distributed in the plant kingdom. Studies have shown CA possesses antithrombotic activity in mouse cerebral arterioles in vivo and inhibits platelet aggregation in vitro. However, little is known regarding the effects of CA on platelet-mediated clot retraction. We investigated the effects of CA on platelet activation and clot retraction in response to thrombin. CA inhibited thrombin-induced platelet aggregation, calcium mobilization, adenosine 1,4,5-tri-phosphate (ATP) release, P-selectin expression and fibrinogen binding to integrin αIIbß3 activation without inducing any cytotoxic effect, and inhibited the phosphorylations of protein kinase B (Akt) and extracellular signal-regulated kinase (ERK) in thrombin-stimulated platelets. In addition, CA enhanced cyclic adenosine monophosphate (cAMP) generation, which led to the phosphorylations of vasodilator-stimulated phosphoprotein (VASP) and inositol trisphosphate (IP3) receptor, and reduced clot retraction without any anticoagulation effect. Dipyridamole, a phosphodiesterase 3 (PDE3) inhibitor, reduced clot retraction, which suggested CA-mediated cAMP generation is the main signaling pathway responsible for its inhibition of clot retraction. Taken together, the findings of the present study suggest that CA may have potential as a therapeutic for the prevention of thrombotic disorders.

Arctigenin attenuates platelet activation and clot retraction by regulation of thromboxane A2 synthesis and cAMP pathway.

Pathophysiological reaction of platelets in the blood vessel is an indispensable part of thrombosis and cardiovascular disease, which is the most common cause of death in the world. In this study, we performed in vitro assays to evaluate antiplatelet activity of arctigenin in human platelets and attempted to identify the mechanism responsible for thromboxane A2 (TXA2) generation, integrin αIIbß3 activation and cAMP pathway. Arctigenin exhibited obvious inhibitory effects on collagen-, thrombin-, and ADP-induced human platelet aggregation, granule secretion, TXA2 generation, integrin αIIbß3 activation, and clot retraction. Additionally, we found that arctigenin attenuated PI3K/Akt/mTOR/GSK-3ß and MAPK signaling pathways, and increased cAMP level. Accordingly, the findings support that arctigenin attenuates thrombotic events through the inhibition of platelet activation and platelet plug formation. Therefore, we suggest that arctigenin may have therapeutic potential as an antiplatelet and antithrombotic agent.