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.

Dasatinib Inhibits Procoagulant and Clot Retracting Activities of Human Platelets.

Tyrosine kinase inhibitors (TKI) such as the BCR-ABL inhibitor dasatinib and nilotinib are highly effective therapies for chronic myeloid leukemia (CML). However, several lines of evidence suggest that dasatinib can induce bleeding which may be due to impaired collagen-induced platelet adhesion, aggregation, and secretion. Sarcoma family kinases (SFK) play central role in the GPVI-induced signaling pathway. We aimed to investigate whether and how dasatinib can modulate SFK-mediated platelet procoagulant activity in a purified system and in dasatinib/nilotinib treated CML patients. In platelet rich plasmas of healthy volunteers, dasatinib dose-dependently reduced convulxin-induced phosphatidylserine exposure and attenuated thrombin formation. Similarly to these changes, integrin activation and clot retraction were also significantly inhibited by 100 nM dasatinib. Platelets isolated from dasatinib treated patients showed a significantly lower phosphatidylserine expression upon convulxin activation compared to premedication levels. In these samples, thrombin generation was significantly slower, and the quantity of formed thrombin was less compared to the trough sample. Western blot analyses showed decreased phosphorylation levels of the C-terminal tail and the activation loop of SFKs upon dasatinib administration. Taken together, these results suggest that dasatinib inhibits the formation of procoagulant platelets via the GPVI receptor by inhibiting phosphorylation of SFKs.

Platycodin D inhibits platelet function and thrombus formation through inducing internalization of platelet glycoprotein receptors.

BACKGROUND: Platycodin D (PD) is one of the major bioactive components of the roots of Platycodon grandiflorum and possesses multiple biological and pharmacological properties, such as antiviral, anti-inflammatory, and anti-cancer activities. However, whether it affects platelet function remains unclear. This study aims to evaluate the role of PD in platelet function and thrombus formation. METHODS: Platelets were treated with PD followed by measuring platelet aggregation, activation, spreading, clot retraction, expression of glycoprotein receptors. Moreover, mice platelets were treated with PD and infused into wild-type mice for analysis of in vivo hemostasis and arterial thrombosis. RESULTS: Platycodin D treatment significantly inhibited platelet aggregation in response to collagen, ADP, arachidonic acid and epinephrine, reduced platelet P-selectin expression, integrin αIIbß3 activation, spreading on fibrinogen as well as clot retraction, accompanied with decreased phosphorylation of Syk and PLCγ2 in collagen-related peptide or thrombin-stimulated platelets. Moreover, PD-treated mice platelets presented significantly impaired in vivo hemostasis and arterial thrombus formation. Interestingly, PD induced internalization of glycoprotein receptors αIIbß3, GPIbα and GPVI. However, GM6001, cytochalasin D, BAPTA-AM and wortmannin did not prevent PD-induced internalization of receptors. CONCLUSIONS: Our study demonstrates that PD inhibits platelet aggregation, activation and impairs hemostasis and arterial thrombosis, suggesting it might be a potent anti-thrombotic drug.

In vitro and in vivo antithrombotic and cytotoxicity effects of ferulic acid.

We discovered recently in vitro and in vivo antithrombotic and cytotoxicity effects of ferulic acid. The cytotoxicity assays showed that ferulic acid (∼300 µg/mL) did not cause any significant toxicity on three cell lines, platelets, leukocytes, and erythrocytes. In vitro assays showed inhibitory effects of ferulic acid on thrombin (THR)- or collagen/epinephrine-stimulated platelet activation by inhibiting platelet aggregation, and decreasing clot retraction activity. The in vitro effect of ferulic acid on THR-stimulated platelet activation was proved by the decrease in the secretion of serotonin from the platelets. The anticoagulant effects of ferulic acid were confirmed by the prolongation of the intrinsic or/and extrinsic pathways and the delay of recalcification time in plasma coagulation. Ferulic acid had antithrombotic effect in acute thromboembolism model in vivo, and decreased the expression of αIIb ß3 /FIB and phosphorylation of AKT in THR-stimulated platelet activation in vivo, and their antithrombotic efficacies hold promise for therapeutic targeting in our ongoing studies.

A novel STAT3 inhibitor negatively modulates platelet activation and aggregation.

The signal transducer and activator of transcription 3 (STAT3) plays a critical role in platelet functions. This study sought to understand the effects of the STAT3 inhibitor SC99 on platelet activation and aggregation. Immunoblotting assays were applied to measure the effects of SC99 on the STAT3 signaling pathway. A ChronoLog aggregometer was used to evaluate platelet aggregation. A flow cytometer was used to evaluate P-selectin expression in the presence of SC99. AlamarBlue and Annexin-V staining were used to evaluate platelet viability and apoptosis, respectively. A fluorescence microscope was applied to analyze platelet spreading. SC99 inhibited the phosphorylation of JAK2 and STAT3 in human platelets but had no effects on the phosphorylation of AKT, p65 or Src, all of which are involved in platelet activation. Further studies revealed that SC99 inhibited human platelet aggregation induced by collagen and thrombin in a dose-dependent manner. SC99 inhibited thrombin-induced P-selectin expression and fibrinogen binding to single platelets. Moreover, SC99 inhibited platelet spreading on fibrinogen and clot retraction mediated by outside-in signaling. SC99 inhibited platelet aggregation in mice but it did not significantly prolong the bleeding time. Taken together, the present study revealed that SC99 inhibited platelet activation and aggregation as a STAT3 inhibitor. This agent can be developed as a promising treatment for thrombotic disorders.

Reduced clot retraction rate and altered platelet energy production in patients with asthma.

OBJECTIVE: Asthma enhances the risk of pulmonary embolism. The mechanism of this phenomenon is unclear. METHODS: We evaluated the kinetics of clot formation, clot retraction rate (CRR), clot volume at 40 min, the rate of lactate production (a marker of aerobic glycolysis in platelets in contracting clots), blood eosinophil count (EOS), nitric oxide in exhaled breath (FENO), and spirometry (FEV1) in 50 healthy controls and in 81 allergic asthmatics (41 subjects with steroid-naïve asthma and 40 with steroid-treated asthma). RESULTS: Thromboelastometry revealed that only steroid-treated asthmatics had slightly activated coagulation. Compared with healthy controls, whole asthmatics demonstrated (p < 0.05) reduced CRR, higher clot volume at 40 minutes, higher FENO, decreased FEV1, elevated EOS, and augmented lactate production in retracting clots. Reduced CRR was observed also in the absence of native plasma. In whole study population (asthmatics and healthy controls), CRR positively correlated with spirometry (rS = 0.668, p = <0.001) and negatively with FENO (rS = -0.543; p < 0.001), EOS (rS = -0.367, p < 0.002), and lactate production (rS = -0.791; p < 0.001). However, in steroid-treated asthmatics, the CRR did not correlate with FENO and EOS. In all study patients lactate production negatively correlated with FEV1 and positively with FENO. CONCLUSION: Collectively, this data is consistent with the hypothesis that, in asthmatics, reactive nitrogen species produced in the lungs may reduce platelet contractility (and CRR) through the diminution of platelet energy production. CRR inhibition would predispose asthmatics to pulmonary embolism.

PDK1 regulates platelet activation and arterial thrombosis.

The effects of phosphoinositide-dependent protein kinase 1 (PDK1), a master kinase in the phosphoinositide 3-kinase/Akt pathway, on platelet activation are unknown. Accordingly, platelet-specific PDK1-deficient mice were characterized to elucidate the platelet-related function(s) of PDK1. We found that PDK1 deficiency caused mild thrombocytopenia. The aggregation of PDK1(-/-) platelets was diminished in response to low levels of thrombin, U46619, and adenosine 5'-diphosphate. Further results demonstrated that PDK1 regulates thrombin-induced platelet activation by affecting αIIbß3-mediated outside-in signaling. This result provided an explanation for the diminished spreading of PDK1(-/-) platelets on immobilized fibrinogen (Fg) and the decreased rate of clot retraction in platelet-rich plasma (PRP) containing PDK1(-/-) platelets. PDK1 deficiency diminished agonist-induced Akt Ser473 phosphorylation and thoroughly abolished Akt Thr308 and Gsk3ß Ser9 phosphorylation in response to agonist treatment and platelet spreading, respectively. A Gsk3ß inhibitor fully restored the aggregation of PDK1(-/-) platelets in response to low levels of thrombin, normal spreading of PDK1(-/-) platelets on Fg, and normal clot retraction in PRP containing PDK1(-/-) platelets. Those results indicated that Gsk3ß is one of the major downstream effectors of PDK1 in thrombin-induced platelet activation and αIIbß3-mediated outside-in signaling. In addition, in vivo data demonstrated that PDK1 is an important regulator in arterial thrombosis formation.

Clot retraction is mediated by factor XIII-dependent fibrin-αIIbß3-myosin axis in platelet sphingomyelin-rich membrane rafts.

Membrane rafts are spatially and functionally heterogenous in the cell membrane. We observed that lysenin-positive sphingomyelin (SM)-rich rafts are identified histochemically in the central region of adhered platelets where fibrin and myosin are colocalized on activation by thrombin. The clot retraction of SM-depleted platelets from SM synthase knockout mouse was delayed significantly, suggesting that platelet SM-rich rafts are involved in clot retraction. We found that fibrin converted by thrombin translocated immediately in platelet detergent-resistant membrane (DRM) rafts but that from Glanzmann's thrombasthenic platelets failed. The fibrinogen γ-chain C-terminal (residues 144-411) fusion protein translocated to platelet DRM rafts on thrombin activation, but its mutant that was replaced by A398A399 at factor XIII crosslinking sites (Q398Q399) was inhibited. Furthermore, fibrin translocation to DRM rafts was impaired in factor XIII A subunit-deficient mouse platelets, which show impaired clot retraction. In the cytoplasm, myosin translocated concomitantly with fibrin translocation into the DRM raft of thrombin-stimulated platelets. Furthermore, the disruption of SM-rich rafts by methyl-ß-cyclodextrin impaired myosin activation and clot retraction. Thus, we propose that clot retraction takes place in SM-rich rafts where a fibrin-αIIbß3-myosin complex is formed as a primary axis to promote platelet contraction.

Distinct roles for Rap1b protein in platelet secretion and integrin αIIbß3 outside-in signaling.

Rap1b is activated by platelet agonists and plays a critical role in integrin α(IIb)ß(3) inside-out signaling and platelet aggregation. Here we show that agonist-induced Rap1b activation plays an important role in stimulating secretion of platelet granules. We also show that α(IIb)ß(3) outside-in signaling can activate Rap1b, and integrin outside-in signaling-mediated Rap1b activation is important in facilitating platelet spreading on fibrinogen and clot retraction. Rap1b-deficient platelets had diminished ATP secretion and P-selectin expression induced by thrombin or collagen. Importantly, addition of low doses of ADP and/or fibrinogen restored aggregation of Rap1b-deficient platelets. Furthermore, we found that Rap1b was activated by platelet spreading on immobilized fibrinogen, a process that was not affected by P2Y(12) or TXA(2) receptor deficiency, but was inhibited by the selective Src inhibitor PP2, the PKC inhibitor Ro-31-8220, or the calcium chelator demethyl-1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis. Clot retraction was abolished, and platelet spreading on fibrinogen was diminished in Rap1b-deficient platelets compared with wild-type controls. The defects in clot retraction and spreading on fibrinogen of Rap1b-deficient platelets were not rescued by addition of MnCl(2), which elicits α(IIb)ß(3) outside-in signaling in the absence of inside-out signaling. Thus, our results reveal two different activation mechanisms of Rap1b as well as novel functions of Rap1b in platelet secretion and in integrin α(IIb)ß(3) outside-in signaling.

Phosphoinositide 3-kinase p110 beta regulates integrin alpha IIb beta 3 avidity and the cellular transmission of contractile forces.

Phosphoinositide (PI) 3-kinase (PI3K) signaling processes play an important role in regulating the adhesive function of integrin alpha(IIb)beta(3), necessary for platelet spreading and sustained platelet aggregation. PI3K inhibitors are effective at reducing platelet aggregation and thrombus formation in vivo and as a consequence are currently being evaluated as novel antithrombotic agents. PI3K regulation of integrin alpha(IIb)beta(3) activation (affinity modulation) primarily occurs downstream of G(i)-coupled and tyrosine kinase-linked receptors linked to the activation of Rap1b, AKT, and phospholipase C. In the present study, we demonstrate an important role for PI3Ks in regulating the avidity (strength of adhesion) of high affinity integrin alpha(IIb)beta(3) bonds, necessary for the cellular transmission of contractile forces. Using knock-out mouse models and isoform-selective PI3K inhibitors, we demonstrate that the Type Ia p110 beta isoform plays a major role in regulating thrombin-stimulated fibrin clot retraction in vitro. Reduced clot retraction induced by PI3K inhibitors was not associated with defects in integrin alpha(IIb)beta(3) activation, actin polymerization, or actomyosin contractility but was associated with a defect in integrin alpha(IIb)beta(3) association with the contractile cytoskeleton. Analysis of integrin alpha(IIb)beta(3) adhesion contacts using total internal reflection fluorescence microscopy revealed an important role for PI3Ks in regulating the stability of high affinity integrin alpha(IIb)beta(3) bonds. These studies demonstrate an important role for PI3K p110 beta in regulating the avidity of high affinity integrin alpha(IIb)beta(3) receptors, necessary for the cellular transmission of contractile forces. These findings may provide new insight into the potential antithrombotic properties of PI3K p110 beta inhibitors.

Monitoring rFVIIa 90 µg kg⁻¹ dosing in haemophiliacs: comparing laboratory response using various whole blood assays over 6 h.

Recombinant FVIIa is a haemostatic agent administered to patients with severe FVIII or FIX deficiency with inhibitors. Although rFVIIa is effective at stopping bleeding, a reliable assay to monitor its effect is lacking. To characterize the pharmacokinetics and global coagulation effects of rFVIIa for 6 h following a IV dose of 90 µg kg⁻¹. Ten non-bleeding subjects with severe FVIII or FIX deficiency were infused with a single-dose of rFVIIa 90 µg k⁻¹ body weight and blood was collected before and at 0.5, 1, 2, 4 and 6 h postdose. Global haemostasis was characterized throughout the study utilizing whole blood analyses (Hemodyne HAS, TEG, ROTEM). The clearance and half-life of factor FVII:C was estimated as 39.0 ± 8.8 mL h⁻¹ kg⁻¹ and 2.1 ± 0.2 h respectively. There was good inter-assay agreement with respect to clot initiation parameters (R, CT and FOT) and these parameters all fell to a mean of approximately 9 min following rFVIIa dosing. The platelet contractile force (PCF) and clot elastic modulus (CEM) were positively correlated to FVII:C (P < 0.0001), and these parameters were dynamic throughout the 6-h period. The MA and MCF did not correlate to FVII:C nor did they significantly change during the study. Prothrombin F1 + 2 significantly increased following rFVIIa dosing (P < 0.001), but remained steady throughout the study. There was no change in D-dimer concentrations over time. The FOT, R and CT characterized clot initiation following rFVIIa dosing. The PCF and CEM were correlated to FVII:C and characterized the dynamics of platelet function and clot strength over the rFVIIa dosing interval. The clinical significance of these findings needs additional study.

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.

RGT, a synthetic peptide corresponding to the integrin beta 3 cytoplasmic C-terminal sequence, selectively inhibits outside-in signaling in human platelets by disrupting the interaction of integrin alpha IIb beta 3 with Src kinase.

Mutational analysis has established that the cytoplasmic tail of the integrin beta 3 subunit binds c-Src (termed as Src in this study) and is critical for bidirectional integrin signaling. Here we show in washed human platelets that a cell-permeable, myristoylated RGT peptide (myr-RGT) corresponding to the integrin beta 3 C-terminal sequence dose-dependently inhibited stable platelet adhesion and spreading on immobilized fibrinogen, and fibrin clot retraction as well. Myr-RGT also inhibited the aggregation-dependent platelet secretion and secretion-dependent second wave of platelet aggregation induced by adenosine diphosphate, ristocetin, or thrombin. Thus, myr-RGT inhibited integrin outside-in signaling. In contrast, myr-RGT had no inhibitory effect on adenosine diphosphate-induced soluble fibrinogen binding to platelets that is dependent on integrin inside-out signaling. Furthermore, the RGT peptide induced dissociation of Src from integrin beta 3 and dose-dependently inhibited the purified recombinant beta 3 cytoplasmic domain binding to Src-SH3. In addition, phosphorylation of the beta 3 cytoplasmic tyrosines, Y(747) and Y(759), was inhibited by myr-RGT. These data indicate an important role for beta 3-Src interaction in outside-in signaling. Thus, in intact human platelets, disruption of the association of Src with beta 3 and selective blockade of integrin alpha IIb beta 3 outside-in signaling by myr-RGT suggest a potential new antithrombotic strategy.

Platelet quality after washing: the effect of storage time before washing.

BACKGROUND: Patients who have experienced anaphylactic transfusion reactions receive washed platelet (PLT) concentrates (PCs) where the plasma has been substituted with a PLT additive solution. This study compares the in vitro quality of PCs washed at the beginning of the storage period (Day 1) to PCs washed at the end of storage (Day 7). STUDY DESIGN AND METHODS: PLTs were prepared by the buffy coat procedure. Two concentrates were pooled and then split to obtain an identical pair of PCs. One of the PCs was washed with T-Sol on Day 1 and the other on Day 7 of storage. Swirling, blood gases, and metabolic variables were analyzed before washing. Analyses of surface expression of CD62P and coagulation by free oscillation rheometry (FOR) were performed before and after washing. RESULTS: pH was acceptable in all PCs. Washing on Days 1 and 7 increased the CD62P surface expression. The FOR variables clotting time and clot retraction were not influenced by washing on either day. Washing resulted in a decrease in the number of PLTs and the decrease was larger on Day 7 compared to Day 1. CONCLUSIONS: PLTs washed on Days 1 and 7 of storage are effected by washing in a similar manner. However, a larger loss of PLTs occurred during washing on Day 7.

A new plasma-adapted hydroxyethyl starch preparation: in vitro coagulation studies.

OBJECTIVE: Preparing hydroxyethyl starch (HES) in a plasma-adapted solution is supposed to improve safety with regard to coagulation. The influence of a new plasma-adapted HES preparation on coagulation was studied. DESIGN: Operator-blinded, randomized study. SETTING: Laboratory in vitro study. PARTICIPANTS: Fifteen healthy young men scheduled for blood donation. INTERVENTIONS: Blood was diluted by 10%, 30%, and 50% using either a plasma-adapted or nonplasma-adapted (prepared in saline solution) potato-derived 6% HES 130/0.42. Only the composition of the solvent of the 2 HES preparations was different. MEASUREMENTS AND MAIN RESULTS: Rotation thromboelastometry (ROTEM; Pentapharm, Munich, Germany) was used to assess changes in coagulation; whole blood aggregometry with 3 inducers was used to assess effects of dilution with HES on platelet function. Clotting time (CT) and clot formation time were significantly prolonged by 30% and 50% dilution, showing significantly longer times in the non-plasma-adapted than in the plasma-adapted HES group (eg, intrinsic CT at the 30% dilution level: plasma-adapted HES 228 +/- 26 seconds [within normal range] v 269 +/- 29 seconds in the nonplasma-adapted HES group). Clot strengthening and clot firmness were significantly reduced by the non-plasma-adapted HES at the 30% dilution level. Platelet aggregation was significantly more reduced by the non-plasma-adapted HES at the 30% and 50% dilution levels. CONCLUSIONS: Dilution with the nonplasma-adapted HES 130/0.42 was associated with more negative effects on thromboelastometry and platelet aggregation than the same HES 130/0.4 dissolved in a plasma-adapted solution. The benefits of using a plasma-adapted modern HES preparation on blood loss and use of blood/blood products in cardiac surgery need to be studied.

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.

d-dimer is a predictor of clot resolution in patients with pulmonary thromboembolism: A retrospective cohort study.

BACKGROUND: This study assessed predictors of pulmonary thromboembolism (PE) resolution and their implications for clinical outcome. METHOD: A total of 150 patients with acute PE diagnosed by computed tomography pulmonary angiography (CTPA) were included. All patients received anticoagulant therapy for 3-6 months and were followed-up for at least 2 years. d-dimer levels in plasma were assayed at the first admission and during follow-up. RESULTS: The rate of CTPA-confirmed PE resolution was 48.67% at 6 months, 68% at 12 months, and 78.67% at 24 months. Thirty-nine patients had recurrent thrombosis after anticoagulation therapy was stopped, whereas 93 patients had complete resolution. The initial d-dimer level positively correlated with the pulmonary artery obstruction index (PAOI) (r = 0.21; P = 0.015), but did not significantly differ between patients experiencing resolution or recurrence. In contrast, the follow-up mean d-dimer level was significantly higher in the recurrent group (P < 0.001), and this level was an independent risk factor for recurrent PE after the termination of anticoagulation treatment (OR 1.003, 95%CI 1.002 to 1.004; P < 0.001). Higher initial thromboembolic burden measured by PAOI was associated with residual thromboemboli (P = 0.004) and recurrence (P = 0.03), but was not an independent risk factor for either. CONCLUSIONS: Elevated d-dimer is an independent risk factor for PE recurrence. A higher initial thromboembolic burden may be associated with unresolved thromboemboli or recurrence.

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.

Mechanisms of venous thrombosis and resolution.

Venous thromboembolism is a significant health care problem in the US. In this review, the unique role of inflammation to the venous thrombotic process is emphasized as well as the potential role of abnormalities of fibrinolytic mechanisms to the thrombotic process. Inflammation influences not only thrombogenesis but also thrombus resolution and vein wall remodeling, and these interactions are also discussed. Knowledge of molecular and immunologic mechanisms for venous thrombosis and its resolution should allow for the future development of targeted therapies.

Morin hydrate inhibits platelet activation and clot retraction by regulating integrin αIIbß3, TXA2, and cAMP levels.

Morin hydrate is an active constituent of Morus alba L, Prunus dulcis, and Cudrania tricuspidata and has been reported to inhibit platelet activation in vivo and in vitro, but no reports have been issued on its regulation of αIIbß3, a platelet-specific integrin and thromboxane A2 (TXA2), positive feedback molecule. In this study, we investigated the anti-platelet activity of morin hydrate in collagen- and thrombin-induced human platelets and attempted to identify the mechanism responsible for integrin αIIbß3 activation and TXA2 generation. Our results demonstrated that morin hydrate (25-100 µM) inhibited collagen- and thrombin-induced platelet aggregation, granule secretion (P-selectin expression, ATP, and serotonin release), calcium mobilization, TXA2 production, integrin αIIbß3 activation, and clot retraction. Additionally, morin hydrate attenuated the phosphorylations of phospholipase Cγ2 (PLCγ2), cytosolic phospholipase A2 (cPLA2), phosphoinositide 3-kinase (PI3K), Akt, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK), and enhanced the phosphorylations of inositol trisphosphate receptor (IP3 receptor) and cyclic adenosine monophosphate (cAMP) generation. However, it had no effect on the coagulation pathway. Taken together, these observations indicate morin hydrate inhibits platelet-mediated thrombosis by down-regulating TXA2 production and integrin αIIbß3 activation, and by upregulating cAMP generation, and thus, inhibits clot retraction. These results suggest morin hydrate may have therapeutic potential as a treatment for platelet-activation-related diseases.