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Introduction: Luteolin inhibits platelet activation and thrombus formation, but the mechanisms are unclear. This study investigated the effects of luteolin on GPVImediated platelet activation in vitro and explored the effect of luteolin on thrombosis, coagulation, and platelet production in vivo. Methods: Washed human platelets were used for aggregation, membrane protein expression, ATP, Ca2+, and LDH release, platelet adhesion/spreading, and clot retraction experiments. Washed human platelets were used to detect collagen and convulxin-induced reactive oxygen species production and endogenous antioxidant effects. C57BL/6 male mice were used for ferric chloride-induced mesenteric thrombosis, collagen-epinephrine induced acute pulmonary embolism, tail bleeding, coagulation function, and luteolin toxicity experiments. The interaction between luteolin and GPVI was analyzed using solid phase binding assay and surface plasmon resonance (SPR). Results: Luteolin inhibited collagen- and convulxin-mediated platelet aggregation, adhesion, and release. Luteolin inhibited collagen- and convulxininduced platelet ROS production and increased platelet endogenous antioxidant capacity. Luteolin reduced convulxin-induced activation of ITAM and MAPK signaling molecules. Molecular docking simulation showed that luteolin forms hydrogen bonds with GPVI. The solid phase binding assay showed that luteolin inhibited the interaction between collagen and GPVI. Surface plasmon resonance showed that luteolin bonded GPVI. Luteolin inhibited integrin αIIbβ3-mediated platelet activation. Luteolin inhibited mesenteric artery thrombosis and collagenadrenergic-induced pulmonary thrombosis in mice. Luteolin decreased oxidative stress in vivo. Luteolin did not affect coagulation, hemostasis, or platelet production in mice. Discussion: Luteolin may be an effective and safe antiplatelet agent target for GPVI. A new mechanism (decreased oxidative stress) for the anti-platelet activity of luteolin has been identified. [ABSTRACT FROM AUTHOR]

Background: Berberine (BBR), a natural product, was reported to inhibit platelet aggregation; however, the molecular mechanisms remain unclear. This study aims to investigate the effects and mechanisms of BBR in inhibiting platelet activation and thrombus formation.Methods: Flow cytometry, immunofluorescence, and Western blot were used to determine the inhibitory effects and mechanisms of BBR and its main metabolite berberrubine (M2) on platelet activation in vitro and ex vivo. Purified integrin αIIbβ3, class I PI3K kit, and molecular docking were used to identify the possible targets of BBR and M2. A carrageenan-induced mouse thrombosis model was used to evaluate the effects of BBR on thrombus formation in vivo.Results:In vitro, BBR and M2 significantly inhibited ADP-induced integrin αIIbβ3 activation, reduced the level of P-selectin on the platelet membrane, and suppressed the binding of fibrinogen to the platelets. In this process, BBR and M2 greatly suppressed the PI3K/Akt pathway and inhibited Rasa3 membrane translocation and Rap1 activation. Furthermore, BBR and M2 selectively inhibited class I PI3Kβ, perhaps through binding to its active site. The activities of BBR were stronger than those of M2. After oral administration, BBR significantly inhibited the PI3K/Akt pathway and Rap1 activation and suppressed ADP-induced platelet activation and carrageenan-induced thrombosis in mice without prolonging bleeding time.Conclusions: We reveal for the first time the possible targets and mechanisms of BBR and M2 in inhibiting platelet activation. Our research may support the future clinical application of BBR as an antiplatelet drug in the prevention or treatment of thrombotic diseases.

Background: Berberine (BBR), a natural product, was reported to inhibit platelet aggregation; however, the molecular mechanisms remain unclear. This study aims to investigate the effects and mechanisms of BBR in inhibiting platelet activation and thrombus formation. Methods: Flow cytometry, immunofluorescence, and Western blot were used to determine the inhibitory effects and mechanisms of BBR and its main metabolite berberrubine (M2) on platelet activation in vitro and ex vivo. Purified integrin αIIbβ3, class I PI3K kit, and molecular docking were used to identify the possible targets of BBR and M2. A carrageenan-induced mouse thrombosis model was used to evaluate the effects of BBR on thrombus formation in vivo. Results: In vitro , BBR and M2 significantly inhibited ADP-induced integrin αIIbβ3 activation, reduced the level of P-selectin on the platelet membrane, and suppressed the binding of fibrinogen to the platelets. In this process, BBR and M2 greatly suppressed the PI3K/Akt pathway and inhibited Rasa3 membrane translocation and Rap1 activation. Furthermore, BBR and M2 selectively inhibited class I PI3Kβ, perhaps through binding to its active site. The activities of BBR were stronger than those of M2. After oral administration, BBR significantly inhibited the PI3K/Akt pathway and Rap1 activation and suppressed ADP-induced platelet activation and carrageenan-induced thrombosis in mice without prolonging bleeding time. Conclusions: We reveal for the first time the possible targets and mechanisms of BBR and M2 in inhibiting platelet activation. Our research may support the future clinical application of BBR as an antiplatelet drug in the prevention or treatment of thrombotic diseases. [ABSTRACT FROM AUTHOR]

Background: Adrenaline is believed to play a role in thrombosis and hemostasis. The complex effect of its clinically relevant concentrations on thrombus formation, coagulation and fibrinolysis in human blood has never been specifically studied.Methods: Confocal microscopy was used to study thrombus formation under flow, exposure of phosphatidylserine (PS) in adhered platelets, to evaluate clots density, and to measure kinetics of fibrin formation and external fibrinolysis under flow. Flow cytometry was utilized to assess PS exposure in non-adhered platelets. Kinetics of clot formation and internal fibrinolysis was evaluated by thromboelastometry. Platelet aggregation was measured by optical aggremometry. Kinetics of clot retraction was assessed by using digital camera.Results: We found that adrenaline (1–10 nM) is able to enhance platelet activation evoked by subthreshold collagen (150 ng/ml), resulting in augmentation of platelet aggregation, thrombus formation under arterial flow conditions, platelet PS exposure, and formation of platelet-fibrin clots. The development of platelet procoagulant response evoked by adrenaline + low collagen was associated with the formation of denser platelet-fibrin clots and the decrease in rate of fibrinolysis despite whether lysis was initiated inside (internal fibrinolysis) or outside the clot (external fibrinolysis). The above phenomena were abolished by the α2-adrenergic receptor antagonist, rauwolscine. Adrenaline-collagen synergism, expressed as PS exposure, was significantly reduced by cyclooxygenase inhibitor (acetylsalicic acid), GPIIb/IIIa receptor blocker (tirofiban), and P2Y12 receptor antagonist (PSB 0739).Conclusion: Clinically relevant concentrations of adrenaline may significantly augment responses of human platelets in the presence of subthreshold concentrations of collagen, which should be considered during therapies involving adrenaline infusion. Routinely used antiplatelet drugs may reduce the prothrombotic state evoked by adrenaline-collagen synergism.

Background: Adrenaline is believed to play a role in thrombosis and hemostasis. The complex effect of its clinically relevant concentrations on thrombus formation, coagulation and fibrinolysis in human blood has never been specifically studied. Methods: Confocal microscopy was used to study thrombus formation under flow, exposure of phosphatidylserine (PS) in adhered platelets, to evaluate clots density, and to measure kinetics of fibrin formation and external fibrinolysis under flow. Flow cytometry was utilized to assess PS exposure in non-adhered platelets. Kinetics of clot formation and internal fibrinolysis was evaluated by thromboelastometry. Platelet aggregation was measured by optical aggremometry. Kinetics of clot retraction was assessed by using digital camera. Results: We found that adrenaline (1–10 nM) is able to enhance platelet activation evoked by subthreshold collagen (150 ng/ml), resulting in augmentation of platelet aggregation, thrombus formation under arterial flow conditions, platelet PS exposure, and formation of platelet-fibrin clots. The development of platelet procoagulant response evoked by adrenaline + low collagen was associated with the formation of denser platelet-fibrin clots and the decrease in rate of fibrinolysis despite whether lysis was initiated inside (internal fibrinolysis) or outside the clot (external fibrinolysis). The above phenomena were abolished by the α2-adrenergic receptor antagonist, rauwolscine. Adrenaline-collagen synergism, expressed as PS exposure, was significantly reduced by cyclooxygenase inhibitor (acetylsalicic acid), GPIIb/IIIa receptor blocker (tirofiban), and P2Y12 receptor antagonist (PSB 0739). Conclusion: Clinically relevant concentrations of adrenaline may significantly augment responses of human platelets in the presence of subthreshold concentrations of collagen, which should be considered during therapies involving adrenaline infusion. Routinely used antiplatelet drugs may reduce the prothrombotic state evoked by adrenaline-collagen synergism. [ABSTRACT FROM AUTHOR]

Although platelets and the coagulation factors are components of the blood system, they become part of and contribute to the tumor microenvironment (TME) not only within a solid tumor mass, but also within a hematogenous micrometastasis on its way through the blood stream to the metastatic niche. The latter basically consists of blood-borne cancer cells which are in close association with platelets. At the site of the primary tumor, the blood components reach the TME via leaky blood vessels, whose permeability is increased by tumor-secreted growth factors, by incomplete angiogenic sprouts or by vasculogenic mimicry (VM) vessels. As a consequence, platelets reach the primary tumor via several cell adhesion molecules (CAMs). Moreover, clotting factor VII from the blood associates with tissue factor (TF) that is abundantly expressed on cancer cells. This extrinsic tenase complex turns on the coagulation cascade, which encompasses the activation of thrombin and conversion of soluble fibrinogen into insoluble fibrin. The presence of platelets and their release of growth factors, as well as fibrin deposition changes the TME of a solid tumor mass substantially, thereby promoting tumor progression. Disseminating cancer cells that circulate in the blood stream also recruit platelets, primarily by direct cell-cell interactions via different receptor-counterreceptor pairs and indirectly by fibrin, which bridges the two cell types via different integrin receptors. These tumor cell-platelet aggregates are hematogenous micrometastases, in which platelets and fibrin constitute a particular TME in favor of the cancer cells. Even at the distant site of settlement, the accompanying platelets help the tumor cell to attach and to grow into metastases. Understanding the close liaison of cancer cells with platelets and coagulation factors that change the TME during tumor progression and spreading will help to curb different steps of the metastatic cascade and may help to reduce tumor-induced thrombosis.

Background: Recent studies indicate that aquaporin (AQP) water channels have a regulatory function in human platelet secretion and in procoagulant response of murine platelets. However, the engagement of AQPs in morphological changes, procoagulant response, and thrombus formation in human blood has never been investigated.Methods: Confocal microscopy was used to study platelet spreading, filopodia formation, ballooning, and thrombus formation under flow. Flow cytometry was utilized to assess platelet phosphatidylserine (PS) exposure and microparticles shedding. Kinetics of clot formation in vitro was evaluated by thromboelastometry. Mouse model of ferric chloride (III) (FeCl3)-induced thrombosis was used to investigate thrombus formation in vivo.Results: We found that chloroauric(III) acid (HAuCl4), a classical AQP inhibitor (10–100 μM), reduced spreading of human platelets on collagen-coated surfaces and inhibited filopodia formation in a fluid phase. Under flow conditions, HAuCl4 (100 μM) attenuated thrombi growth on collagen, platelet secretion, and PS exposure. Thrombus formation was restored by the addition of exogenous adenosine diphosphate (ADP). Collagen-evoked platelet procoagulant response (evaluated as PS exposure, shedding of microparticles, platelet-dependent thrombin generation, and membrane ballooning) was distinctly reduced by HAuCl4 (25–200 μM), as well as the dynamics of clot formation. In mouse model of thrombosis, reduction of surface of PS-positive cells within thrombus was observed in the presence of HAuCl4 (1–10 mg/kg).Conclusion: These results suggest that in human platelets AQPs are crucial for agonist-evoked morphological changes, thrombus formation under flow, and in development of procoagulant response. Antithrombotic effect in vivo suggests that nontoxic inhibitors of AQPs may be considered as potential candidates for a novel class of antiplatelet drugs.

Objective: A rare case of thromboembolic cerebral infarction due to carotid web in a patient with congenital protein C deficiency is reported.Case Presentation: A patient in her 40's with left-side hemiparesis was transferred to our hospital under continuous intravenous injection of heparin. Magnetic resonance angiography demonstrated occlusion of the right middle cerebral artery (MCA). Conventional angiography revealed recanalization of the right MCA and a carotid web at the origin of the right internal carotid artery. Ultrasound scan of the carotid artery on the 19th day revealed thrombus formation on the distal portion of the carotid web. We performed carotid artery stenting to prevent thrombus formation by suppressing the carotid web to the vessel wall and by regulating the turbulent flow. The patient had no recurrence of stroke under-anticoagulation with warfarin during the 2-year follow-up period.Conclusion: To our knowledge, this is the first report in which an immediate thrombus formation on the carotid web was observed in a patient with congenital protein C deficiency. In a case of acute ischemic stroke with carotid web, especially when congenital coagulopathy such as protein C deficiency is suspected, careful follow-up with ultrasound imaging should be performed.

Background : Recent studies indicate that aquaporin (AQP) water channels have a regulatory function in human platelet secretion and in procoagulant response of murine platelets. However, the engagement of AQPs in morphological changes, procoagulant response, and thrombus formation in human blood has never been investigated. Methods : Confocal microscopy was used to study platelet spreading, filopodia formation, ballooning, and thrombus formation under flow. Flow cytometry was utilized to assess platelet phosphatidylserine (PS) exposure and microparticles shedding. Kinetics of clot formation in vitro was evaluated by thromboelastometry. Mouse model of ferric chloride (III) (FeCl3)-induced thrombosis was used to investigate thrombus formation in vivo. Results : We found that chloroauric(III) acid (HAuCl4), a classical AQP inhibitor (10–100 μM), reduced spreading of human platelets on collagen-coated surfaces and inhibited filopodia formation in a fluid phase. Under flow conditions, HAuCl4 (100 μM) attenuated thrombi growth on collagen, platelet secretion, and PS exposure. Thrombus formation was restored by the addition of exogenous adenosine diphosphate (ADP). Collagen-evoked platelet procoagulant response (evaluated as PS exposure, shedding of microparticles, platelet-dependent thrombin generation, and membrane ballooning) was distinctly reduced by HAuCl4 (25–200 μM), as well as the dynamics of clot formation. In mouse model of thrombosis, reduction of surface of PS-positive cells within thrombus was observed in the presence of HAuCl4 (1–10 mg/kg). Conclusion : These results suggest that in human platelets AQPs are crucial for agonist-evoked morphological changes, thrombus formation under flow, and in development of procoagulant response. Antithrombotic effect in vivo suggests that nontoxic inhibitors of AQPs may be considered as potential candidates for a novel class of antiplatelet drugs. [ABSTRACT FROM AUTHOR]

Objective: A rare case of thromboembolic cerebral infarction due to carotid web in a patient with congenital protein C deficiency is reported. Case Presentation: A patient in her 40's with left-side hemiparesis was transferred to our hospital under continuous intravenous injection of heparin. Magnetic resonance angiography demonstrated occlusion of the right middle cerebral artery (MCA). Conventional angiography revealed recanalization of the right MCA and a carotid web at the origin of the right internal carotid artery. Ultrasound scan of the carotid artery on the 19th day revealed thrombus formation on the distal portion of the carotid web. We performed carotid artery stenting to prevent thrombus formation by suppressing the carotid web to the vessel wall and by regulating the turbulent flow. The patient had no recurrence of stroke under-anticoagulation with warfarin during the 2-year follow-up period. Conclusion: To our knowledge, this is the first report in which an immediate thrombus formation on the carotid web was observed in a patient with congenital protein C deficiency. In a case of acute ischemic stroke with carotid web, especially when congenital coagulopathy such as protein C deficiency is suspected, careful follow-up with ultrasound imaging should be performed. [ABSTRACT FROM AUTHOR]

HD1 and HD22 are two of the most-studied aptamers binding to thrombin exosite I and exosite, respectively. To complete of their pharmacological profiles, the effects of HD1 and HD22 on thrombin-, ristocetin-, and collagen-induced human platelet aggregation, on thrombin generation and fibrin formation in human plasma, as well as on thrombus formation in human whole blood under flow conditions were assessed. The dissociation constants for HD1 and HD22 complexes with thrombin in simulated plasma ionic buffer were also evaluated. HD1 was more potent than HD22 in terms of inhibiting thrombin-induced platelet aggregation in platelet-rich plasma (PRP; 0.05–3 μM) and in washed platelets (WPs; 0.005–3 μM): approximately 8.31% (±6.99% SD) and 89.53% (±11.38% SD) for HD1 (0.5 μM) and HD22 (0.5 μM), respectively. Neither HD1 nor HD22 (3 μM) did influence platelets aggregation induced by collagen. Both of them inhibited ristocetin-induced aggregation in PRP. Surprisingly, HD1 and HD22 aptamers (3 μM) potentiated ristocetin-induced platelet aggregation in WP. HD1 reduced thrombin generation in a concentration-dependent manner [ETP at 3 μM: 1677.53 ± 55.77 (nM⋅min) vs. control 2271.71 ± 423.66 (nM⋅min)], inhibited fibrin formation (lag time at 3 μM: 33.70 min ± 8.01 min vs. control 7.91 min ± 0.91 min) and reduced thrombus formation under flow conditions [AUC30 at 3 μM: 758.30 ± 344.23 (kPa⋅min) vs. control 1553.84 ± 118.03 (kPa⋅min)]. HD22 (3 μM) also delayed thrombin generation but increased the thrombin peak. HD22 (3 μM) shortened the lag time of fibrin generation (5.40 min ± 0.26 min vs. control 7.58 min ± 1.14 min) but did not modify thrombus formation (3, 15 μM). Kd values for the HD1 complex with thrombin was higher (257.8 ± 15.0 nM) than the Kd for HD22 (97.6 ± 2.2 nM). In conclusion, HD1 but not HD22 represents a potent anti-thrombotic agent, confirming the major role of exosite I in the action of thrombin. HD22 aptamer blocking exosite II displays weaker anti-platelet and anti-coagulant activity, with surprising activating effects on thrombin and fibrin generation most likely induced by HD22-induced allosteric changes in thrombin dynamic structure.

HD1 and HD22 are two of the most-studied aptamers binding to thrombin exosite I and exosite, respectively. To complete of their pharmacological profiles, the effects of HD1 and HD22 on thrombin-, ristocetin-, and collagen-induced human platelet aggregation, on thrombin generation and fibrin formation in human plasma, as well as on thrombus formation in human whole blood under flow conditions were assessed. The dissociation constants for HD1 and HD22 complexes with thrombin in simulated plasma ionic buffer were also evaluated. HD1 was more potent than HD22 in terms of inhibiting thrombin-induced platelet aggregation in platelet-rich plasma (PRP; 0.05–3 μM) and in washed platelets (WPs; 0.005–3 μM): approximately 8.31% (±6.99% SD) and 89.53% (±11.38% SD) for HD1 (0.5 μM) and HD22 (0.5 μM), respectively. Neither HD1 nor HD22 (3 μM) did influence platelets aggregation induced by collagen. Both of them inhibited ristocetin-induced aggregation in PRP. Surprisingly, HD1 and HD22 aptamers (3 μM) potentiated ristocetin-induced platelet aggregation in WP. HD1 reduced thrombin generation in a concentration-dependent manner [ETP at 3 μM: 1677.53 ± 55.77 (nM⋅min) vs. control 2271.71 ± 423.66 (nM⋅min)], inhibited fibrin formation (lag time at 3 μM: 33.70 min ± 8.01 min vs. control 7.91 min ± 0.91 min) and reduced thrombus formation under flow conditions [AUC30 at 3 μM: 758.30 ± 344.23 (kPa⋅min) vs. control 1553.84 ± 118.03 (kPa⋅min)]. HD22 (3 μM) also delayed thrombin generation but increased the thrombin peak. HD22 (3 μM) shortened the lag time of fibrin generation (5.40 min ± 0.26 min vs. control 7.58 min ± 1.14 min) but did not modify thrombus formation (3, 15 μM). K d values for the HD1 complex with thrombin was higher (257.8 ± 15.0 nM) than the K d for HD22 (97.6 ± 2.2 nM). In conclusion, HD1 but not HD22 represents a potent anti-thrombotic agent, confirming the major role of exosite I in the action of thrombin. HD22 aptamer blocking exosite II displays weaker anti-platelet and anti-coagulant activity, with surprising activating effects on thrombin and fibrin generation most likely induced by HD22-induced allosteric changes in thrombin dynamic structure. [ABSTRACT FROM AUTHOR]