Fibrin Clot Retraction Research Articles

Heat stress–induced platelet dysfunction is associated with loss of fibrinogen and is improved by fibrinogen supplementation

IntroductionHeatstroke is a critical heat-related condition characterized by coagulopathy and multiple organ dysfunction. One of the most severe complications of heatstroke is disseminated intravascular coagulation. This condition manifests as excessive clot formation and bleeding that are primarily due to platelet depletion and dysfunction. Fibrinogen plays a crucial role in hemostasis because it links integrin αIIbβ3 on adjacent platelets, thereby promoting the platelet activation and aggregation necessary for clot formation. However, reduced fibrinogen levels may impair the formation of the initial platelet plug and increase the risk of bleeding. The current study explored the effect of fibrinogen on platelet dysfunction in a heatstroke model. Materials and methodsMale Wistar rats were subjected to heat stress, and subsequent changes in hemodynamic, biochemical, and coagulation parameters were analyzed. Platelet viability, aggregation, adhesion, spreading and fibrin clot retraction were assessed. ResultsThe rats with heatstroke exhibited a variety of clinical symptoms, including hypotension, tachycardia, multiple organ dysfunction, and coagulopathy. Platelet viability in the heatstroke group was comparable to that in the healthy control group. However, the heatstroke group exhibited significant reductions in plasma fibrinogen levels and platelet aggregation, adhesion, spreading, and fibrin clot retraction. Notably, fibrinogen supplementation markedly augmented the aggregation responses of platelets in the heatstroke group. The impairment of platelet adhesion, spreading, and fibrin clot retraction in the rats with heatstroke was partially ameliorated by fibrinogen supplementation. ConclusionsAn early use of fibrinogen replacement may serve as a therapeutic intervention to alleviate platelet hyporeactivity and prevent the complications in patients with heatstroke.

Opposing Roles for the α Isoform of the Catalytic Subunit of Protein Phosphatase 1 in Inside-Out and Outside-In Integrin Signaling in Murine Platelets.

Platelet activation during hemostasis and thrombosis is facilitated by agonist-induced inside-out and integrin αIIbβ3-initiated outside-in signaling via protein kinases and phosphatases. Pharmacological inhibitor studies suggest that the serine/threonine protein phosphatase 1 (PP1) promotes platelet activation. However, since phosphatase inhibitors block all the isoforms of the catalytic subunit of PP1 (PP1c), the role of specific PP1c isoform in platelet signaling remains unclear. Here, we employed a platelet-specific PP1cα-/- mice to explore the contribution of a major PP1 isoform in platelet functions. Loss of PP1cα moderately decreased activation of integrin αIIbβ3, binding of soluble fibrinogen, and aggregation to low-dose thrombin, ADP, and collagen. In contrast, PP1cα-/- platelets displayed increased adhesion to immobilized fibrinogen, fibrin clot retraction, and thrombus formation on immobilized collagen. Mechanistically, post-fibrinogen engagement potentiated p38 mitogen-activated protein kinase (MAPK) activation in PP1cα-/- platelets and the p38 inhibitor blocked the increased integrin-mediated outside-in signaling function. Tail bleeding time and light-dye injury-induced microvascular thrombosis in the cremaster venules and arterioles were not altered in PP1cα-/- mice. Thus, PP1cα displays pleiotropic signaling in platelets as it amplifies agonist-induced signaling and attenuates integrin-mediated signaling with no impact on hemostasis and thrombosis.

Garcinol acts as a novel integrin αIIbβ3 inhibitor in human platelets

AimsPlatelet activation plays a central role in arterial thrombosis. Platelets are activated by adhesive proteins (i.e., collagen) or soluble agonists (i.e., thrombin), the respective receptor-specific signaling cause inside–out signaling, leading to the binding of fibrinogen to integrin αIIbβ3. This binding triggers outside–in signaling, resulting in platelet aggregation. Garcinol, a polyisoprenylated benzophenone, is extracted from the fruit rind of Garcinia indica. Although garcinol exhibits considerable bioactivities, few studies have investigated the effect of garcinol on platelet activation. Main methodsAggregometry, immunoblotting, flow cytometer, confocal microscopic analysis, fibrin clot retraction, animal studies such as fluorescein-induced platelet plug formation in mesenteric microvessels, acute pulmonary thromboembolism, and tail bleeding time were performed in this study. Key findingsThis study indicates that garcinol inhibited platelet aggregation stimulated by collagen, thrombin, arachidonic acid, and U46619. Garcinol reduced integrin αIIbβ3 inside–out signaling, including ATP release; cytosolic Ca2+ mobilization; P-selectin expression; and Syk, PLCγ2/PKC, PI3K/Akt/GSK3β, MAPKs, and NF-κB activation stimulated by collagen. Garcinol directly inhibited integrin αIIbβ3 activation by interfering with FITC–PAC-1 and FITC-triflavin by collagen. Additionally, garcinol affected integrin αIIbβ3-mediated outside–in signaling, such as decreasing platelet adhesion and the single-platelet spreading area; suppressing integrin β3, Src, FAK, and Syk phosphorylation on immobilized fibrinogen; and inhibiting thrombin-stimulated fibrin clot retraction. Garcinol substantially reduced mortality caused by pulmonary thromboembolism and prolonged the occlusion time of thrombotic platelet plug formation without extending bleeding time in mice. SignificanceThis study identified that garcinol, a novel antithrombotic agent, acts as a naturally occurring integrin αIIbβ3 inhibitor.

Investigation of Blood Coagulation Using Impedance Spectroscopy: Toward Innovative Biomarkers to Assess Fibrinogenesis and Clot Retraction.

This study focused on a coagulation assessment based on the novel technique of blood-impedance-magnitude measurement. With the impedance characterization of recalcified human blood, it was possible to identify two significative biomarkers (i.e., measurable indicators) related to fibrin formation (1st marker) and clot retraction (2nd marker). The confocal microscopy of clotting blood provided a complete visual analysis of all the events occurring during coagulation, validating the significance of the impedance biomarkers. By analyzing the impedance phase angle (Φ) of blood during coagulation, as well as those of the clot and serum expelled after retraction, it was possible to further clarify the origin of the 2nd marker. Finally, an impedance-magnitude analysis and a rotational thromboelastometry test (ROTEM®) were simultaneously performed on blood sampled from the same donor; the results pointed out that the 1st marker was related to clotting time. The developed technique gives rise to a comprehensive and evolutive insight into coagulation, making it possible to progressively follow the whole process in real time. Moreover, this approach allows coagulation to be tested on any materials’ surface, laying the ground for new studies related to contact coagulation, meaning, thrombosis occurring on artificial implants. In a near future, impedance spectroscopy could be employed in the material characterization of cardiovascular prostheses whose properties could be monitored in situ and/or online using effective biomarkers.

Antiplatelet Activity of Tetramethylpyrazine via Regulation of the P2Y12 Receptor Downstream Signaling Pathway

Background Tetramethylpyrazine (TMP) is an alkaloid in Chinese herbal medicine, which possesses antiplatelet activity. TMP inhibits platelet activation in many ways. The platelet P2Y12 receptor for adenosine 5′ diphosphate (ADP) plays a central role in platelet function, hemostasis, and thrombosis. Here, we investigated the inhibitory effect of TMP on P2Y12 receptor-related platelet function. Methods The inhibitory potential of TMP was assessed using agonist-induced platelet aggregation, flow cytometric analysis of CD62p expression, PAC-1 activation, and fibrin clot retraction. After the P2Y12 receptor-related signaling pathway was inhibited using the blocker, platelet activation was studied by platelet aggregation, CD62p expression, and PAC-1 activation. The secretion of cyclic adenosine monophosphate (cAMP) was measured using enzyme-linked immunosorbent assay (ELISA), and the expression of signaling pathway protein, phosphorylation of vasodilator-stimulated phosphoprotein, and phosphorylation of Akt were investigated using western blotting. The release of platelet inflammatory mediators was measured using ELISA. Results TMP had an antiplatelet effect by inhibiting ADP-induced aggregation, P-selectin secretion, and glycoprotein (GP) IIb/IIIa expression and reducing the release of atherosclerotic-related inflammatory mediators (sCD40L and IL-1β). TMP decreased the area of clot retraction, reflecting inhibition of GPIIb/IIIa activation. TMP inhibited adenosine diphosphate-induced platelet activation via increased cAMP production, VASPser157 phosphorylation, and Akt dephosphorylation. Conclusion TMP selectively inhibits ADP-induced platelet activation via P2Y12 receptor-related signaling pathways.

Agonist-Induced Signaling and Integrin-Mediated Function in Murine Platelets Is Distinctly Regulated By the α Isoform of the Catalytic Subunit of Protein Phosphatase 1

Platelet activation at the site of injury is promoted by agonist-induced inside-out and integrin αIIbβ3-initiated outside-in signaling. Our understanding of the agonist and integrin mediated signaling pathways has largely been driven by studies that have examined the role of protein kinases. As models of signal transduction have been refined to include protein phosphorylation and protein dephosphorylation events, the contribution of the protein phosphatases to platelet signaling and function have garnered fresh attention. This is particularly evident for the catalytic subunit of serine/threonine protein phosphatase 1 (PP1c), wherein, its role in platelet biology has been previously assessed with pharmacological agents like calyculin A and okadaic acid. Although such chemical inhibitors represent a useful tool, it lacks specificity to discriminate the different isoforms of PP1c. PP1cα represents a major platelet expressed isoform and its contribution to platelet function is not known. To explore the potential role of PP1cα isoform in platelet signaling, we generated a platelet specific PP1cα -/- mice by crossing the PP1cα floxed mice with a platelet factor 4 (PF4) cre mice. Immunoblotting studies from PP1cα -/- mice confirmed that PP1cα was lost in platelets but not in lymphocytes. A compensatory upregulation of the PP1cβ but not PP1cγ1 isoform was noticed in PP1cα -/- platelets. Loss of PP1cα decreased platelet aggregation and soluble fibrinogen binding to low dose thrombin, thromboxane analog and convulxin, suggesting that PP1cα promotes agonist-induced inside-out signaling. In contrast, PP1cα -/- platelets displayed enhanced adhesiveness to immobilized fibrinogen and increased fibrin clot retraction, processes that are dependent on integrin-mediated outside-in signaling. In a microfluidic perfusion system, whole blood from PP1cα -/- mice displayed increased thrombus formation on collagen. p38 mitogen activated protein kinase (MAPK) was activated in PP1cα -/- platelets, and p38 inhibitor blocked the increased integrin-mediated PP1cα -/- platelet functions. These observations suggest that PP1cα suppress integrin-mediated function in part by decreasing the activation of p38 MAPK. Thus, unlike the conclusion gathered from generic phosphatase inhibitors that PP1c promotes platelet function, our studies using a genetic approach indicate that PP1cα contributes to platelet signaling in a fashion that is specific to the mode of signaling. Specifically, PP1cα promotes platelet agonist-induced inside-out signaling and suppress integrin-mediated outside-in signaling. This observation suggests that chemical activators of PP1cα isoform could serve as a potential antithrombotic agent. DisclosuresNo relevant conflicts of interest to declare.

The Antithrombotic Agent Pterostilbene Interferes with Integrin αIIbβ3-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 αIIbβ3 activation through interfering with PAC-1 binding stimulated by collagen. In addition, pterostilbene affected integrin αIIbβ3-mediated outside-in signaling, such as integrin β3, 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 αIIbβ3-mediated inside-out and outside-in signaling, suggesting that pterostilbene can serve as a therapeutic agent for thromboembolic disorders.

Modulation of human platelet activation and in vivo vascular thrombosis by columbianadin: regulation by integrin \u03b1IIb\u03b23 inside-out but not outside-in signals

BackgroundColumbianadin (CBN) is one of the main coumarin constituents isolated from Angelica pubescens. The pharmacological value of CBN is well demonstrated, especially in the prevention of several cancers and analgesic activity. A striking therapeutic target for arterial thrombosis is inhibition of platelet activation because platelet activation significantly contributes to these diseases. The current study examined the influence of CBN on human platelet activation in vitro and vascular thrombotic formation in vivo.MethodsAggregometry, immunoblotting, immunoprecipitation, confocal microscopic analysis, fibrin clot retraction, and thrombogenic animals were used in this study.ResultsCBN markedly inhibited platelet aggregation in washed human platelets stimulated only by collagen, but was not effective in platelets stimulated by other agonists such as thrombin, arachidonic acid, and U46619. CBN evidently inhibited ATP release, intracellular ([Ca2+]i) mobilization, and P-selectin expression. It also inhibited the phosphorylation of phospholipase C (PLC)γ2, protein kinase C (PKC), Akt (protein kinase B), and mitogen-activated protein kinases (MAPKs; extracellular signal-regulated kinase [ERK] 1/2 and c-Jun N-terminal kinase [JNK] 1/2, but not p38 MAPK) in collagen-activated platelets. Neither SQ22536, an adenylate cyclase inhibitor, nor ODQ, a guanylate cyclase inhibitor, reversed the CBN-mediated inhibition of platelet aggregation. CBN had no significant effect in triggering vasodilator-stimulated phosphoprotein phosphorylation. Moreover, it markedly hindered integrin αIIbβ3 activation by interfering with the binding of PAC-1; nevertheless, it had no influences on integrin αIIbβ3-mediated outside-in signaling such as adhesion number and spreading area of platelets on immobilized fibrinogen as well as thrombin-stimulated fibrin clot retraction. Additionally, CBN did not attenuate FITC-triflavin binding or phosphorylation of proteins, such as integrin β3, Src, and focal adhesion kinase, in platelets spreading on immobilized fibrinogen. In experimental mice, CBN increased the occlusion time of thrombotic platelet plug formation.ConclusionThis study demonstrated that CBN exhibits an exceptional activity against platelet activation through inhibition of the PLCγ2-PKC cascade, subsequently suppressing the activation of Akt and ERKs/JNKs and influencing platelet aggregation. Consequently, this work provides solid evidence and considers that CBN has the potential to serve as a therapeutic agent for the treatment of thromboembolic disorders.

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.

Suppression of Human Platelet Activation via Integrin αIIbβ3 Outside-In Independent Signal and Reduction of the Mortality in Pulmonary Thrombosis by Auraptene.

Auraptene is the most abundant coumarin derivative from plants. The pharmacological value of this compound has been well demonstrated, especially in the prevention of cancer and neurodegenerative diseases. Platelet activation is a major factor contributing to arterial thrombosis. Thus, this study evaluated the influence of auraptene in platelet aggregation and thrombotic formation. Auraptene inhibited platelet aggregation in human platelets stimulated with collagen only. However, auraptene was not effective in inhibiting platelet aggregation stimulated with thrombin, arachidonic acid, and U46619. Auraptene also repressed ATP release, [Ca2+]i mobilization, and P-selectin expression. Moreover, it markedly blocked PAC-1 binding to integrin αIIbβ3. However, it had no influence on properties related to integrin αIIbβ3-mediated outside-in signaling, such as the adhesion number, spreading area of platelets, and fibrin clot retraction. Auraptene inhibited the phosphorylation of Lyn-Fyn-Syk, phospholipase Cγ2 (PLCγ2), protein kinase C (PKC), Akt, and mitogen-activated protein kinases (MAPKs; extracellular-signal-regulated kinase (ERK1/2), and c-Jun N-terminal kinase (JNK1/2), but not p38 MAPK). Neither SQ22536, an adenylate cyclase inhibitor, nor ODQ, a guanylate cyclase inhibitor, reversed the auraptene-mediated inhibition of platelet aggregation. Auraptene reduced mortality caused by adenosine diphosphate (ADP)-induced pulmonary thromboembolism. In conclusion, this study provides definite evidence that auraptene signifies a potential therapeutic agent for preventing thromboembolic disorders.

Platelet-like particles dynamically stiffen fibrin matrices and improve wound healing outcomes.

Native platelets perform several critical functions within the context of wound healing, including participating in initial hemostasis and interacting with fibrin at the wound site to induce clot retraction. Platelet depletion or dysfunction due to trauma or disease can inhibit robust wound healing responses. There has been a focus recently on developing synthetic, non-immunogenic platelet mimetic technologies for the purpose of augmenting hemostatic responses in cases of deficient native platelet functionality. Here we describe the application of synthetic platelet-like particles (PLPs), capable of recapitulating the deformable platelet body and fibrin specificity found in native platelets, to enhance healing outcomes. We first demonstrate PLPs mimic activated platelet morphology and induce fibrin clot retraction. During clot retraction, native platelets generate forces within a fibrin network to stiffen the fibrin matrix; therefore, we hypothesized that our PLPs will likewise be able to stiffen provisional fibrin matrices. Due to previous studies indicating that increased matrix stiffness is linked to increased cellular migration, we further hypothesize that PLP-mediated fibrin stiffening will enhance cell migration and improve healing outcomes within in vitro and in vivo models of wound healing. PLPs were found to enhance fibroblast migration in in vitro models of early wound healing and enhance healing outcomes in an in vivo murine model of wound healing. These studies demonstrate the utility of PLPs for enhancing wound repair and also provide insight into the role of native platelet-mediated clot retraction in wound healing.

Wdr-1 is essential for F-actin interaction with focal adhesions in platelets.

: Wdr-1, an actin interacting protein, enhances cofilin's capacity to accelerate depolymerization of F-actin filaments. Wdr-1-deficient mice have impaired hemostasis due to defective inside-out integrin signaling in platelets. Here, we studied the role of Wdr-1 on outside-in signaling necessary for retraction of the clot and platelet spreading. Outside-in signaling was assessed by fibrin clot retraction assay and by adhesion and spreading of unstimulated platelets on fibrinogen substrate. The spatial distribution of actin, cofilin-1 and Wdr-1 were determined by immunofluorescence microscopy. Interaction of F-actin with focal adhesion kinase was assessed in dual-color confocal images and by immunoblotting of F-actin filaments. Clot retraction is markedly impaired in Wdr-1-deficient platelets. Wdr-1-deficient platelets adhere and spread poorly on fibrinogen substrate compared with wild-type controls. In resting platelets, Wdr-1 is colocalized with cofilin-1 in cortical actin. Following platelets spreading on fibrinogen substrate, Wdr-1 translocates to the cytoskeleton in association with cofilin-1. In Wdr-1-deficient platelets, cofilin-1 is aberrantly localized throughout the cytoplasm and there is no significant change following adhesion to fibrinogen substrate. The actin filaments formed upon spreading on fibrinogen are mostly in the periphery of the platelets and does not traverse the cytoplasm. Furthermore, there is diminished colocalization of actin filaments with focal adhesion kinase. These studies show that Wdr-1 is essential for the localization of cofilin-1 to the platelet membrane skeleton. F-actin fails to attach to focal adhesions resulting in defective reorganization of actin filaments necessary for platelet spreading and clot retraction.

Antiplatelet and antithrombotic effects of cordycepin-enriched WIB-801CE from Cordyceps militaris ex vivo, in vivo, and in vitro.

BackgroundA species of the fungal genus Cordyceps has been used as a complementary and alternative medicine of traditional Chinese medicine, and its major component cordycepin and cordycepin-enriched WIB-801CE are known to have antiplatelet effects in vitro. However, it is unknown whether they have also endogenous antiplatelet and antithrombotic effects. In this study, to resolve these doubts, we prepared cordycepin-enriched WIB-801CE, an ethanol extract from Cordyceps militaris-hypha, then evaluated its ex vivo, in vivo, and in vitro antiplatelet and antithrombotic effects.MethodsEx vivo effects of WIB-801CE on collagen- and ADP-induced platelet aggregation, serotonin release, thromboxane A2 (TXA2) production and its associated activities of enzymes [cyclooxygenase-1 (COX-1), TXA2 synthase (TXAS)], arachidonic acid (AA) release and its associated phosphorylation of phospholipase Cβ3, phospholipase Cγ2 or cytosolic phospholipase A2, mitogen-activated protein kinase (MAPK) [p38 MAPK, extracellular signal-regulated kinase (ERK)], and blood coagulation time in rats were investigated. In vivo effects of WIB-801CE on collagen plus epinephrine-induced acute pulmonary thromboembolism, and tail bleeding time in mice were also inquired. In vitro effects of WIB-801CE on cytotoxicity, and fibrin clot retraction in human platelets, and nitric oxide (NO) production in RAW264.7 cells or free radical scavenging activity were studied.ResultsCordycepin-enriched WIB-801CE inhibited ex vivo platelet aggregation, TXA2 production, AA release, TXAS activity, serotonin release, and p38 MAPK and ERK2 phosphorylation in collagen- and ADP-activated rat platelets without affecting blood coagulation. Furthermore, WIB-801CE manifested in vivo inhibitory effect on collagen plus epinephrine-induced pulmonary thromboembolism mice model. WIB-801CE inhibited in vitro NO production and fibrin clot retraction, but elevated free radical scavenging activity without affecting cytotoxicity against human platelets.ConclusionWIB-801CE inhibited collagen- and ADP-induced platelet activation and its associated thrombus formation ex vivo and in vivo. These were resulted from down-regulation of TXA2 production and its related AA release and TXAS activity, and p38 MAPK and ERK2 activation. These results suggest that WIB-801CE has therapeutic potential to treat platelet activation-mediated thrombotic diseases in vivo.

A Novel Interaction of the Catalytic Subunit of Protein Phosphatase 2A with the Adaptor Protein CIN85 Suppresses Phosphatase Activity and Facilitates Platelet Outside-in αIIbβ3 Integrin Signaling

The transduction of signals generated by protein kinases and phosphatases are critical for the ability of integrin αIIbβ3 to support stable platelet adhesion and thrombus formation. Unlike kinases, it remains unclear how serine/threonine phosphatases engage the signaling networks that are initiated following integrin ligation. Because protein-protein interactions form the backbone of signal transduction, we searched for proteins that interact with the catalytic subunit of protein phosphatase 2A (PP2Ac). In a yeast two-hybrid study, we identified a novel interaction between PP2Ac and an adaptor protein CIN85 (Cbl-interacting protein of 85 kDa). Truncation and alanine mutagenesis studies revealed that PP2Ac binds to the P3 block ((396)PAIPPKKPRP(405)) of the proline-rich region in CIN85. The interaction of purified PP2Ac with CIN85 suppressed phosphatase activity. Human embryonal kidney 293 αIIbβ3 cells overexpressing a CIN85 P3 mutant, which cannot support PP2Ac binding, displayed decreased adhesion to immobilized fibrinogen. Platelets contain the ∼85 kDa CIN85 protein along with the PP2Ac-CIN85 complex. A myristylated cell-permeable peptide derived from residues 395-407 of CIN85 protein (P3 peptide) disrupted the platelet PP2Ac-CIN85 complex and decreased αIIbβ3 signaling dependent functions such as platelet spreading on fibrinogen and thrombin-mediated fibrin clot retraction. In a phospho-profiling study P3 peptide treated platelets also displayed decreased phosphorylation of several signaling proteins including Src and GSK3β. Taken together, these data support a role for the novel PP2Ac-CIN85 complex in supporting integrin-dependent platelet function by dampening the phosphatase activity.

Compaction of fibrin clots reveals the antifibrinolytic effect of factor XIII

Essentials Factor XIIIa inhibits fibrinolysis by forming fibrin-fibrin and fibrin-inhibitor cross-links. Conflicting studies about magnitude and mechanisms of inhibition have been reported. Factor XIIIa most strongly inhibits lysis of mechanically compacted or retracted plasma clots. Cross-links of α2-antiplasmin to fibrin prevent the inhibitor from being expelled from the clot. Background Although insights into the underlying mechanisms of the effect of factor XIII on fibrinolysis have improved considerably in the last few decades, in particular with the discovery that activated FXIII (FXIIIa) cross-links α2 -antiplasmin to fibrin, the topic remains a matter of debate. Objective To elucidate the mechanisms of the antifibrinolytic effect of FXIII. Methods and Results Platelet-poor plasma clot lysis, induced by the addition of tissue-type plasminogen activator, was measured in the presence or absence of a specific FXIIIa inhibitor. Both in a turbidity assay and in a fluorescence assay, the FXIIIa inhibitor had only a small inhibitory effect: 1.6-fold less tissue-type plasminogen activator was required for 50% clot lysis in the presence of the FXIIIa inhibitor. However, when the plasma clot was compacted by centrifugation, the FXIIIa inhibitor had a strong inhibitory effect, with 7.7-fold less tissue-type plasminogen activator being required for 50% clot lysis in the presence of the FXIIIa inhibitor. In both experiments, the effects of the FXIIIa inhibitor were entirely dependent on the cross-linking of α2 -antiplasmin to fibrin. The FXIIIa inhibitor reduced the amount of α2 -antiplasmin present in the compacted clots from approximately 30% to < 4%. The results were confirmed with experiments in which compaction was achieved by platelet-mediated clot retraction. Conclusions Compaction or retraction of fibrin clots reveals the strong antifibrinolytic effect of FXIII. This is explained by the cross-linking of α2 -antiplasmin to fibrin by FXIIIa, which prevents the plasmin inhibitor from being fully expelled from the clot during compaction/retraction.

CEACAM2 positively regulates integrin αIIbβ3-mediated platelet functions

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is an Ig-ITIM superfamily member that regulates integrin αIIbβ3 function. We hypothesized that its twin protein, CEACAM2, exerts a similar physiologic role in murine platelets. CEACAM2-deficient mice (Cc2−/−) displayed prolonged tail bleeding times and increased volume of blood loss. Cc2−/− platelets have moderate integrin αIIbβ3-mediated functional defects with impaired kinetics of platelet spreading on fibrinogen and type I collagen and delayed kinetics in the retraction of fibrin clots in vitro. This functional integrin αIIbβ3 defect could not be attributed to altered integrin αIIbβ3 expression. Cc2−/− platelets displayed normal ‘inside-out’ signaling properties as demonstrated by normal agonist-induced binding of soluble fluorescein isothiocyanate (FITC)-fibrinogen and JON/A antibody binding. This data provides direct evidence that disruption of CEACAM2 induces a moderate integrin αIIbβ3-mediated platelet function defect, and that CEACAM2 is essential to maintain a normal integrin αIIbβ3-mediated platelet function.

Myr-RKEFAK Peptide Selectively Regulates Outside-in Signaling Transduction-related Functions in Human Platelets

To study the effect of interaction of the talin rod domain integrin binding site 2 with integrin β3 on platelet signal transduction. A peptide that mimics the membrane proximal α helix 6 residues R724 KEFAK729 of the integrin β3 cytoplasmic tails was designed and synthesized, to which the myristoylation was covalently linked to the N-terminal of the peptide enabling membrane penetration. The effects of myr-RKEFAK peptide on the typical platelet outside-in signaling ovent (stable adhesion and spreading on immobilized fibrinogen, aggregation, fibrin clot retraction) and inside-out signaling events (soluble fibrinogen binding) were tested. myr-RKEFAK peptide dose-dependently inhibited platelet stable adhesion and spreading on immobilized fibrinogen, irreversible aggregation, as well as fibrin clot retraction, but not soluble fibrinogen binding and reversible phase of platelet aggregation. The cell-penetrating peptide myr-RKEFAK causes an inhibitory effect on integrin β3 outside-in signaling-regulated platelets functions, but did not affect inside-out signaling-regulated platelets functions.

The β isoform of the catalytic subunit of protein phosphatase 2B restrains platelet function by suppressing outside‐in αIIbβ3 integrin signaling

Calcium-dependent signaling mechanisms play a critical role in platelet activation. Unlike calcium-activated protease and kinase, the contribution of calcium-activated protein serine/threonine phosphatase in platelet activation is poorly understood. To assess the role of catalytic subunit of protein phosphatase 2B (PP2B) or calcineurin in platelet function. Here, we showed that an increase in PP2B activity was associated with agonist-induced activation of human and murine platelets. Pharmacological inhibitors of the catalytic subunit of protein phosphatase 2B (PP2B-A) such as cyclosporine A or tacrolimus (FK506) potentiated aggregation of human platelets. Murine platelets lacking the β isoform of PP2B-A (PP2B-Aβ(-/-) ) displayed increased aggregation with low doses of agonist concentrations. Loss of PP2B-Aβ did not affect agonist-induced integrin αII b β3 inside-out signaling, but increased basal Src activation and outside-in αII b β3 signaling to p38 mitogen-activated protein kinase (MAPK), with a concomitant enhancement in platelet spreading on immobilized fibrinogen and greater fibrin clot retraction. Fibrinogen-induced increased p38 activation in PP2B-Aβ(-/-) platelets were blocked by Src inhibitor. Both PP2B-Aβ(-/-) platelets and PP2B-Aβ-depleted human embryonal kidney 293 αII b β3 cells displayed increased adhesion to immobilized fibrinogen. Filamin A, an actin crosslinking phosphoprotein that is known to associate with β3 , was dephosphorylated on Ser(2152) in fibrinogen-adhered wild-type but not in PP2B-Aβ(-/-) platelets. In a FeCl3 injury thrombosis model, PP2B-Aβ(-/-) mice showed decreased time to occlusion in the carotid artery. These observations indicate that PP2B-Aβ by suppressing outside-in αII b β3 integrin signaling limits platelet response to vascular injury.

Insights into platelet-based control of coagulation

The coagulation process is activated by tight control mechanisms, in which platelets play prominent and unique roles. In thrombosis and hemostasis, activated platelets regulate the coagulation system in various ways: by exposing a phosphatidylserine surface for thrombin formation, by supporting fibrin formation, and by regulating the retraction of a fibrin clot. In this review we discuss the involvement of platelet receptors, other membrane proteins, downstream signaling proteins, cytoskeleton-linked proteins and plasma proteins in these procoagulant functions. Studies with both genetically modified mice and pharmacological inhibitors indicate that, for collagen-adhered platelets, in part common signaling pathways lead to phosphatidylserine exposure, generation of thrombin and fibrin, and retraction of the fibrin clot. However, prolonged Ca(2+) elevation leads to thrombin generation, whereas integrin-dependent signaling stimulates fibrin clot retraction. Contact-dependent signaling pathways, triggered by homotypic platelet-platelet interactions, act in particular via the integrin route.

Disulfide bond exchanges in integrins αIIbβ3 and αvβ3 are required for activation and post-ligation signaling during clot retraction

Integrin αIIbβ3 mediates platelet adhesion, aggregation and fibrin clot retraction. These processes require activation of αIIbβ3 and post-ligation signaling. Disulfide bond exchanges are involved in αIIbβ3 and αvβ3 activation. In order to investigate the role of integrin activation and disulfide bond exchange during αIIbβ3- and αvβ3-mediated clot retraction, we co-expressed in baby hamster kidney cells wild-type (WT) human αIIb and WT or mutated human β3 that contain single or double cysteine substitutions disrupting C523-C544 or C560-C583 bonds. Flow cytometry was used to measure surface expression and activation state of the integrins. Time-course of fibrin clot retraction was examined. Cells expressed WT or mutated human αIIbβ3 as well as chimeric hamster/human αvβ3. The αIIbβ3 mutants were constitutively active and the thiol blocker dithiobisnitrobenzoic acid (DTNB) did not affect their activation state. WT cells retracted the clot and addition of αvβ3 inhibitors decreased the retraction rate. The active mutants and WT cells activated by anti-LIBS6 antibody retracted the clot faster than untreated WT cells, particularly in the presence of αvβ3 inhibitor. DTNB substantially inhibited clot retraction by WT or double C523S/C544S mutant expressing cells, but minimally affected single C523S, C544S or C560S mutants. Anti-LIBS6-enhanced clot retraction was significantly inhibited by DTNB when added prior to anti-LIBS6. Both αIIbβ3 and αvβ3 contribute to clot retraction without prior activation of the integrins. Activation of αIIbβ3, but not of αvβ3 enhances clot retraction. Both αIIbβ3 activation and post-ligation signaling during clot retraction require disulfide bond exchange.