Platelet Procoagulant Activity Research Articles

Platelet Activation via Shear Stress Exposure Induces a Differing Pattern of Biomarkers of Activation versus Biochemical Agonists

Implantable cardiovascular therapeutic devices, while hemodynamically effective, remain limited by thrombosis. A driver of device-associated thrombosis is shear-mediated platelet activation (SMPA). Underlying mechanisms of SMPA, as well as useful biomarkers able to detect and discriminate mechanical versus biochemical platelet activation, are poorly defined. We hypothesized that SMPA induces a differing pattern of biomarkers compared with biochemical agonists. Gel-filtered human platelets were subjected to mechanical activation via either uniform constant or dynamic shear; or to biochemical activation by adenosine diphosphate (ADP), thrombin receptor-activating peptide 6 (TRAP-6), thrombin, collagen, epinephrine, or arachidonic acid. Markers of platelet activation (P-selectin, integrin αIIbβ3 activation) and apoptosis (mitochondrial membrane potential, caspase 3 activation, and phosphatidylserine externalization [PSE]) were examined using flow cytometry. Platelet procoagulant activity was detected by chromogenic assay measuring thrombin generation. Contribution of platelet calcium flux in SMPA was tested employing calcium chelators, ethylenediaminetetraacetic acid (EDTA), and BAPTA-AM. Platelet exposure to continuous shear stress, but not biochemical agonists, resulted in a dramatic increase of PSE and procoagulant activity, while no integrin αIIbβ3 activation occurred, and P-selectin levels remained barely elevated. SMPA was associated with dissipation of mitochondrial membrane potential, but no caspase 3 activation was observed. Shear-mediated PSE was significantly decreased by chelation of extracellular calcium with EDTA, while intracellular calcium depletion with BAPTA-AM had no significant effect. In contrast, biochemical agonists ADP, TRAP-6, arachidonic acid, and thrombin were potent inducers of αIIbβ3 activation and/or P-selectin exposure. This differing pattern of biomarkers seen for SMPA for continuous uniform shear was replicated in platelets exposed to dynamic shear stress via circulation through a ventricular assist device-propelled circulatory loop. Elevated shear stress, but not biochemical agonists, induces a differing pattern of platelet biomarkers-with enhanced PSE and thrombin generation on the platelet surface. This differential biomarker phenotype of SMPA offers the potential for early detection and discrimination from that mediated by biochemical agonists.

Yin and Yang of MCS-Related Coagulopathy: Shear Stress Promotes Platelet Prothrombosis and Microparticle Generation While Inducing Integrin Downregulation and Decreased Aggregability

Mechanical circulatory support (MCS) has emerged as a lifesaving therapy for patients with advanced and end-stage heart failure. Sadly, MCS therapy is limited by a paradoxical coagulopathy accompanied by both thrombosis and bleeding. We hypothesized that shear-mediated alterations of platelet hemostatic function, i.e. integrin expression, procoagulant activity, and aggregation, mechanistically drive MCS-related coagulopathy. Human platelets were exposed to shear stress in a hemodynamic shearing device (30-70 dyn/cm2, 10 min). Integrin αIIbβ3 & GPIb surface expression/activation, phosphatidylserine externalization (PSE) and microparticles (MPs) were quantified by flow cytometry. Platelet procoagulant activity was verified by thrombin generation assay. Platelet aggregation was measured by optical aggregometry. Shear stress exposure induced notable PSE, simultaneously promoting thrombin generation on platelets and MP. Paradoxically, shear-mediated platelet activation (SMPA) did not induce integrin αIIbβ3 activation. Moreover, high shear (70 dyn/cm2) prevented activation and upregulation of αII2bβ3 mediated by biochemical agonists. Aggregometry findings further validate flow cytometry results, as high shear did not promote aggregation, rather decreased ADP- and TRAP-6 induced aggregation. Likewise, SMPA led to the evident reduction of GPIb surface expression, as indicated by significant drop of GPIb+ platelet number and their fluorescence intensity (73.5 ± 2.9% & 953.1 ± 101.0 AU vs. 94.3 ± 0.7% & 1620 ± 72.4 AU for intact platelets; M ± SEM, ANOVA, p ≤ 0.05). Shear-mediated decrease of GPIb expression along with evident augmentation of GPIb- & αIIbβ3-positive MPs (3.3 ± 0.6% & 12.5 ± 2.8%, respectively) supports integrin shedding as an operative mechanism. SMPA promotes platelet PSE and procoagulant MP generation, thus facilitating thrombin formation and driving prothrombosis. Yet, shear stress did not promote platelet aggregation, rather impaired agonist-induced platelet aggregation via integrin αIIbβ3 and GPIb shedding from the platelets into MPs. This dichotomic effect of shear on platelets mechanistically defines the paradoxical prothrombotic state with a concomitant propensity of bleeding in MCS patients.

Phagocytosis by endothelial cells inhibits procoagulant activity of platelets of essential thrombocythemia in vitro

BackgroundEssential thrombocythemia (ET) is characterized by thrombocytosis with increased platelet number and persistent activation. The mechanisms of thrombosis and the fate of these platelets are not clear. The aim of the present study is to explore the phagocytosis of platelets of ET patients by endothelial cells (ECs) in vitro and its relevance to the procoagulant activity (PCA). MethodsPhosphatidylserine (PS) exposure on platelets was detected by flow cytometry. Phagocytosis of the platelets by ECs was performed using flow cytometry, confocal microscopy, and electron microscopy. The PCA of platelets was evaluated by coagulation time and purified coagulation complex assays. ResultsThe PS exposure on platelets in ET patients is higher than that in healthy controls. The PS‐exposed platelets are highly procoagulant and lactadherin reduced 80% of the PCA by blockade of PS. When cocultured, the platelets of ET patients were sequestered by ECs in a time‐dependent fashion. Lactadherin enhanced phagocytosis by bridging the PS on activated platelets and the integrin αvβ3 on ECs, and P‐selectin played at least a partial role in this process. Furthermore, factor Xa and prothrombinase activity of PS‐exposed platelets were decreased after incubation with ECs. ConclusionOur results suggest that phagocytic clearance of platelets by ECs occurs in ET patients, thus representing a novel mechanism to remove activated platelets from the circulation; lactadherin and phagocytosis could cooperatively limit the thrombophilia in ET patients.

Gene of the issue: ANO6 and Scott Syndrome

Scott Syndrome is a very rare inhibited bleeding disorder characterized by an isolated deficit in procoagulant activity in platelets and other blood cells, caused by a lack of phosphatidylserine (P...

Mechanism of Pulmonary Thrombosis in Hemolytic Disorders

Hemolysis is a hallmark of various inherited or acquired blood disorders including sickle cell disease, thalassemia, paroxysmal nocturnal hemoglobinuria, thrombotic thrombocytopenic purpura and hemolytic-uremic syndrome. Epidemiological evidence suggests that in situ pulmonary thrombosis is a major pathological event contributing to cardiopulmonary morbidities affecting patients with hemolytic disorders. Intravascular hemolysis promotes the release of erythrocyte-derived damage associated molecular patterns (eDAMPs) molecules such as cell free hemoglobin (Hb), heme and adenosine diphosphate (ADP). Although these eDAMPS may activate platelets, promote endothelial dysfunction and drive sterile thrombo-inflammation, the pathogenesis of pulmonary thrombosis in hemolytic disorders and the role of platelets in this process remains unclear. Intravascular hemolysis in C57BL/6 (WT) mice was induced by intravenous (IV) administration of deionized water (H2O). Alternatively, eDAMPs or tissue derived DAMPs relevant to hemolysis or endothelial injury were administered IV to WT mice. The pulmonary microcirculation was visualized using quantitative intravital fluorescence lung microscopy (qFILM). Fluorochrome-conjugated anti-mouse CD49b Ab and dextran were administered IV for in vivo staining of circulating platelets and visualization of blood vessels, respectively. Time series of qFILM images were collected at baseline and post IV administration of agonists to assess pulmonary thrombosis. IV administration of H2O led to arrival of platelet-rich thrombi in the pulmonary arterioles that occluded the arteriolar bottle-necks located at the junction of pulmonary arterioles and capillaries leading to their transient obstruction. DAMPs such as ADP, collagen, thrombin and tissue factor also triggered dose-dependent PT in mice. Identical to IV H2O, DAMPs triggered pulmonary thrombosis also involved entrapment of platelet-rich thrombi in the arteriolar bottle-necks, which led to ischemia in the pulmonary arteriole and the down-stream capillaries. Interestingly, pretreatment with IV heparin (300 U/kg) prevented TF-induced but not ADP-triggered pulmonary thrombosis in mice. In contrast, IV administration of αIIbβ3 receptor inhibitor eptifibatide (10 mg/kg) completely abrogated TF-, collagen and ADP-dependent PT. Our current findings provide the first real-time in vivo visual evidence establishing that intravascular hemolysis directly induces pulmonary thrombosis, which involves entrapment of platelet-rich thrombi in the pre-capillary pulmonary arterioles. Our results are the first to identify that platelet activation and procoagulant activity contribute to pathogenesis of hemolysis induced pulmonary thrombosis. Disclosures Ragni: OPKO: Research Funding; Bioverativ/Sanofi: Other: Advisory Board, Research Funding; Shire/Takeda: Other: Advisory Board, Research Funding; Sangamo: Research Funding; Spark Therapeutics: Other: Advisory Board, Research Funding; Alnylam/Sanofi: Other: Advisory Board, Research Funding; BioMarin: Other: Advisory Board, Research Funding. Neal:Janssen Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; CSL Behring: Membership on an entity's Board of Directors or advisory committees. Gladwin:Bayer Pharmaceuticals: Other: Co-investigator; United Therapeutics: Patents & Royalties: Co-inventor on an NIH government patent for the use of nitrite salts in cardiovascular diseases ; Globin Solutions, Inc: Patents & Royalties: Provisional patents for the use of recombinant neuroglobin and heme-based molecules as antidotes for CO poisoning.

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.

Fibronectin and its soluble EDA-FN isoform as biomarkers for inflammation and sepsis.

Fibronectin (FN) is a widely distributed glycoprotein which is present in different bodily fluids, on the surface of cells and in the extracellular matrix (ECM). It plays roles in various processes, including cell adhesion, migration, growth, proliferation, and tissue repair. Fibronectin exists in 2 forms: a soluble, inactive molecule, called plasma FN (pFN), which is synthesized by hepatocytes in the liver, and an insoluble cellular form (cFN), which is produced locally by different types of cells and is a key component of the ECM. Fibrinogen fibrils ensure structural support for cell adhesion and promote cell migration, proliferation and apoptosis. Additionally, FN controls the availability of growth factors. The plasma form of FN is a crucial component of the fibrin clot in the early wound-healing response, while the cellular form of FN supports efficient platelet adhesion, activation, aggregation, and procoagulant activity. Alternative splicing of the FN gene results in the generation of protein variants which contain the additional isoforms - extra domain A of FN (EDA) and extra domain A of FN (EDB); these are associated with, e.g., tissue remodeling, fibroblast differentiation, inflammation, and tumor progression. Fibronectin also serves as a target for a large number of bacterial proteins, and as part of a 3-component bridge (FN, integrin and FN-binding proteins - FnBPs) it contributes to bacterial colonization of endothelial and epithelial cells. Fibronectin has been identified in sepsis in humans as a negative acute-phase protein, and a low level of FN seems to be a marker of a poor prognosis for a patient. Here, the role of FN in inflammatory processes and sepsis is presented.

PF797 APAC, A DUAL ANTIPLATELET AND ANTICOAGULANT ‐ TOWARDS A LOCAL, VASCULAR TARGETING ANTITHROMBOTIC

Background:APAC, is developed as a mimic of heparin proteoglycans, and has unique antiplatelet and anticoagulant action (collagen and thrombin) in several in vitro and in vivo models (Lassila & Jouppila, Semin Thromb Hemost, 2014). APAC as the only anticoagulant in blood inhibits both platelet and fibrin accumulation on collagen and tissue factor under a high shear rate (1000 1/s), and retains primary adhesion (Chen et al. Thromb Res, 2018). Moreover, APAC protects kidneys from ischemic reperfusion injury (Tuuminen et al. Clin Exp Nephrol 2017). APAC is eliminated via kidneys and liver as unfractionated heparin.Aims:We present APAC and its antithrombotic data on platelet aggregation and global coagulation in vitro, vascular targeting and antithrombotic role in vivo, including toxicology margins.Methods:1. Blood was collected from healthy volunteers. APAC was studied in human platelet aggregation in response to collagen and ristocetin in whole blood (Multiplate), platelet‐rich plasma (PRP) and in rotational thromboelastometry (ROTEM) compared with citrated (3.2%) blood and spiking. APAC's antiplatelet efficacy with acetylsalicylic acid (ASA) was estimated in collagen‐, arachidonic acid (AA)‐ or ADP‐induced platelet aggregation in PRP or whole blood, 2. Porcine arterio‐venous fistula of femoral arterial and venous anastomosis sites and balloon denudation of iliac and carotid arteries were exposed to biotin‐labelled APAC to study APAC binding and localization. 3. Single and repeated dose toxicology of rodents and monkeys was assessed.Results:APAC dose‐dependently attenuates platelet aggregation to collagen and ristocetin, unlike heparin, in citrated whole blood at 150 μg/mL by 58 ± 15% (n = 6) (mean ± SD) and by 25 ± 2%, respectively. In PRP, inhibition of collagen‐induced aggregation was 55 ± 31% at 1 μg/mL APAC and 85 ± 11% at 30 μg/mL APAC (n = 9). APAC alone, also dose‐dependently attenuated aggregation and ROTEM variables in whole blood. In ROTEM, APAC alone (at 3–16 μg/mL) reduces intrinsic pathway of coagulation (INTEM), and acts as a broad platelet‐dependent procoagulant. ASA dose‐dependently inhibited collagen‐induced platelet aggregation, clearly potentiated by APAC requiring up to 4‐fold higher collagen concentration to induce aggregation in PRP. In turn, ADP‐ or AA‐induced aggregation was unaffected. APAC co‐localized with Von Willebrand Factor and laminin in porcine arterial injuries, but not with intact endothelium, which stained positive for PECAM or podocalyxin. Upon targeting vessels, APAC locally decreased platelet accumulation and overt thrombosis in a rat model of anastomosis, but retained at anastomosis sites despite high shear blood flow. Single and multiple (7–14 day) doses (3–20 mg/kg in monkeys and rodents) indicate wide safety without APAC accumulation in the toxicology program.Summary/Conclusion:APAC, inhibits collagen‐ and ristocetin‐, but not ADP‐induced, platelet aggregation in citrated blood and PRP, and synergizes with ASA. In citrated whole blood, and if used as a sole anticoagulant, APAC globally attenuates platelet procoagulant activity and fibrin elasticity. With APAC's unique profile, local integration and inhibition of platelet‐mediated thrombosis may benefit vascular interventions.

A major interspecies difference in the ionic selectivity of megakaryocyte Ca2+-activated channels sensitive to the TMEM16F inhibitor CaCCinh-A01

TMEM16F is a surface membrane protein critical for platelet procoagulant activity, which exhibits both phospholipid scramblase and ion channel activities following sustained elevation of cytosolic Ca2+. The extent to which the ionic permeability of TMEM16F is important for platelet scramblase responses remains controversial. To date, only one study has reported the electrophysiological properties of TMEM16F in cells of platelet/megakaryocyte lineage, which observed cation-selectivity within excised patch recordings from murine marrow-derived megakaryocytes. This contrasts with reports using whole-cell recordings that describe this channel as displaying either selectivity for anions or being relatively non-selective amongst the major physiological monovalent ions.We have studied TMEM16F expression and channel activity in primary rat and mouse megakaryocytes and the human erythroleukemic (HEL) cell line that exhibits megakaryocytic surface markers. Immunocytochemical analysis was consistent with surface TMEM16F expression in cells from all three species. Whole-cell recordings in the absence of K+-selective currents revealed an outwardly rectifying conductance activated by a high intracellular Ca2+ concentration in all three species. These currents appeared after 5–6 minutes and were blocked by CaCCinh-A01, properties typical of TMEM16F. Ion substitution experiments showed that the underlying conductance was predominantly Cl–-permeable in rat megakaryocytes and HEL cells, yet non-selective between monovalent anions and cations in mouse megakaryocytes. In conclusion, the present study further highlights the difference in ionic selectivity of TMEM16F in platelet lineage cells of the mouse compared to other mammalian species. This provides additional support for the ionic “leak” hypothesis that the scramblase activity of TMEM16F does not rely upon its ability to conduct ions of a specific type.

Extracellular mitochondria released from traumatized brains induced platelet procoagulant activity.

Coagulopathy often develops soon after acute traumatic brain injury and its cause remains poorly understood. We have shown that injured brains release cellular microvesicles that disrupt the endothelial barrier and induce consumptive coagulopathy. Morphologically intact extracellular mitochondria accounted for 55.2% of these microvesicles, leading to the hypothesis that these extracellular mitochondria are metabolically active and serve as a source of oxidative stress that activates platelets and renders them procoagulant. In testing this hypothesis experimentally, we found that the extracellular mitochondria purified from brain trauma mice and those released from brains subjected to freeze-thaw injury remained metabolically active and produced reactive oxygen species. These extracellular mitochondria bound platelets through the phospholipid-CD36 interaction and induced α-granule secretion, microvesiculation, and procoagulant activity in an oxidant-dependent manner, but failed to induce aggregation. These results define an extracellular mitochondria-induced and redox-dependent intermediate phenotype of platelets that contribute to the pathogenesis of traumatic brain injury-induced coagulopathy and inflammation.

P2Y12 Receptor Antagonists: Do They Really Inhibit Shear-Mediated Platelet Activation within MCS?

Purpose Shear-mediated platelet activation (SMPA) by supra-physiological blood flow found within MCS is a main driver and contributor of device-related thrombosis. Thus, antiplatelet drugs targeting the main pathways of platelet activation are routinely prescribed. We have shown, that aspirin and GP IIbIIIa blockers have limited ability in mitigating SMPA, and do NOT protect platelets from integrin shedding and microparticle (MP) generation. In contrast, ticagrelor, a direct P2Y12 purinergic receptor antagonist, drastically inhibits platelet prothrombinase activity induced by high shear. The aim of the study was to define the role of the P2Y12 pathway in SMPA and evaluate the effect of P2Y12 antagonists on platelet procoagulant activity and MP generation induced by high shear stress. Methods The following P2Y12 antagonists were used: clopidogrel active metabolite (10 µM), cangrelor (1 µM) and ticagrelor (1 µM). After 10 min incubation with the agent, human platelets were exposed to continuous high shear stress (70 dyn/cm2, 10 min) in a hemodynamic shearing device. Platelet procoagulant activity was assessed using the annexin V binding assay, detecting procoagulant surface exposure, and platelet activity state (PAS) assay measuring thrombin generation. MPs were quantified by flow cytometry as CD41+/AnV+ particles distinguished from platelets by their FS/SS. Results Ticagrelor and cangrelor showed similar inhibitory effect, consistently decreasing both platelet procoagulant activity and microparticle generation induced by shear stress. The number of AnV+ platelets, AnV+ MPs, and PAS levels were decreased by 26%, 27%, and 30%, respectively, as compared with non-treated controls subjected to shear. Clopidogrel active metabolite, known for its low affinity to the targeted receptor, reduced PAS by 15%, while annexin V binding was diminished in more than 10 times for platelets and 2 times for MPs, indicating a significant reduction of platelet procoagulant surface exposure but limited inhibition of thrombin generation. Conclusion P2Y12 purinergic receptor antagonists inhibit shear-mediated platelet procoagulant surface formation and microparticle generation, both contributors in overall platelet prothrombotic activity induced by high shear stress within MCS. Our results suggest that P2Y12 pathway is involved in the regulation of SMPA.

Platelets compensate for poor thrombin generation in type 3 von Willebrand disease

In type 3 von Willebrand disease (VWD3), the most severe form with absent von Willebrand factor (VWF), the bleeding phenotype is variable. Platelet contribution to the hemostatic defect in VWD3 calls upon further studies. We investigated the contribution of platelets to in vitro thrombin generation (TG) and platelet procoagulant activity in VWD3. TG was assessed by calibrated automated thrombogram (CAT) in platelet-poor (PPP) and –rich plasma (PRP) from 9 patients before and in 6 patients also 30 min after receiving their regular VWF therapy. Responsiveness of PPP to FVIII and protein S was also investigated. TG data were compared with routine laboratory variables, rotational thromboelastometry (ROTEM) and platelet expression of P-selectin and phosphatidylserine in flow cytometry. Compared with healthy controls, TG was markedly decreased in VWD3 PPP (peak thrombin was 16% of normal median), but not in PRP (77% of normal median) (p = 0.002). Six out of nine patients (67%) were high responders in their platelet P-selectin, and 5/9 (56%) in phosphatidylserine expression. Replacement therapy improved TG in PPP, while in PRP TG only modestly increased or was unaffected. In PPP, FVIII levels associated with TG and in vitro FVIII-supplemented TG inclined up to threefold. Conversely, a FVIII inhibitory antibody reduced plasma TG in all, but especially in patients with remnant FVIII levels. Inhibition of protein S improved plasma TG, particularly at low FVIII levels. ROTEM failed to detect VWD3.In VWD3, TG is reduced in PPP and regulated by FVIII and protein S, but TG is close to normal in PRP. VWD3 platelets seem to compensate for the FVIII-associated reduction in TG by their exposure of P-selectin and phosphatidylserine.

Successful renal denervation decreases the platelet activation status in hypertensive patients.

To determine whether renal denervation (RDN) in hypertensive patients affects the platelet activation status. We investigated the effect of RDN on the platelet activation status in 41 hypertensive patients undergoing RDN. Ambulatory blood pressure (BP), plasma sympathetic neurotransmitter Neuropeptide Y, and platelet activation markers were measured at baseline, at 3 months, and 6 months after RDN. RDN significantly decreased BP at 3 months (150.6 ± 11.3/80.9 ± 11.4 mmHg to 144.7 ± 12.0/77.1 ± 11.1 mmHg; P < 0.01) and at 6 months (144.3 ± 13.8/78.3 ± 11.1 mmHg; P < 0.01). Plasma levels of the sympathetic neurotransmitter Neuropeptide Y, an indicator of sympathetic nerve activity, were significantly decreased at 3 months (0.29 ± 0.11 ng/mL to 0.23 ± 0.11 ng/mL; P < 0.0001) and at 6 months (0.22 ± 0.12 ng/mL; P < 0.001) after RDN. This was associated with a reduction in platelet membrane P-selectin expression (3 months, P < 0.05; 6 months, P < 0.05), soluble P-selectin (6 months, P < 0.05), circulating numbers of platelet-derived extracellular vesicles (EVs) (3 months, P < 0.001; 6 months, P < 0.01), and phosphatidylserine expressing EVs (3 months, P < 0.001; 6 months, P < 0.0001), indicative of a reduction in platelet activation status and procoagulant activity. Only patients who responded to RDN with a BP reduction showed inhibition of P-selectin expression at 3 months (P < 0.05) and 6 months (P < 0.05) as well as reduction of glycoprotein IIb/IIIa activation at 3 months (P < 0.05). Notably, 13 patients who took aspirin did not show significant reduction in platelet P-selectin expression following RDN. Our results imply a connection between the sympathetic nervous system and the platelet activation status and provide a potential mechanistic explanation by which RDN can have favourable effects towards reducing cardiovascular complications.

Procoagulant Activity of Platelets and Tissue Factor Microparticles in Behçets Disease

Background: Microparticles are found in all body fluids including peripheral blood. They play an important role in blood clot formation and increased cell aggregation. Aim of this work: Was to evaluate the level of circulating platelet microparticles (PMP) and tissue factor microparticles (TFMP) in Behcet`s disease and to assess the relation of these procoagulant markers and Behcet’s disease activity. Patients and methods: This study included 100 persons divided to three groups; Behcet’s disease (BD) group (40) patients, systemic lupus (SLE) group (40) patients as a comparative and control group (20) persons. Platelet-free plasma was ultra-centrifuged. PMPs and TFMPs were identified and enumerated with flow cytometry using anti CD61PE and CD142Percp antibodies. D-dimer was measured by ELISA. Results: There was statistically significant increase of PMP (CD61), tissue factor MP (CD142), CD61-MFI and CD142-MFI in Behcet`s group in comparison to Control group (p˂0.001). There was statistically significant increase of PMP and tissue factor MP in Behcet`s when compared to Lupus group (p˂0.001), while CD61/CD142, CD61- MFI and CD142-MFI revealed no significant difference. D-dimer was significantly increased during Behcet`s and SLE disease activity (p=0.036 & p=0.021 respectively). Active Behcet`s disease had significant increase of both CD61 and tissue factor expressing PMP (CD61/CD142) when compared to patients with inactive state (p˂0.001 & p=0.004 respectively), while CD142 showed no significant difference (p=0.38). In Behcet`s group, there was significant positive correlation between CD61 and each of CD61/CD142 and D-dimer (p=0.001). Conclusion: PMP and TF MP are significantly elevated in BD in comparison to SLE. PMP, PMP expressing TF and D-dimer are markers of disease activity in both BD and SLE, while ESR and CRP are not. PMP is positively correlated to TF expressing PMP and D-dimer in BD denoting enhanced prothrombotic state. PMP, PMP expressing TF and D-dimer are characters of ANCA positive BD.

Procoagulant Activity of Platelets and Tissue Factor Microparticles in Behçets Disease

Procoagulant Activity of Platelets and Tissue Factor Microparticles in Behçets Disease

The Role of Exposed Phosphatidylserine on Microparticles and Blood Cells in Coagulant Activity in Acute Kidney Injury Patients on Continuous Renal Replacement Therapy

Introduction: Serving as the preferred treatment for patients with acute kidney injury (AKI), continuous renal replacement therapy (CRRT) is mostly interrupted by clotting caused by procoagulant state despite of persistent improvements in anticoagulant technology, reducing therapy time, enhancing cost and resulting in blood lost. Due to hemodynamics disorders and foreign material circuit, blood cells undergo definite activation. However, considered as markers of cell activation, the role of phosphatidylserine (PS) and microparticles (MPs) in the hypercoagulability remains largely unexplored. The aim of this study is to measure PS exposure on blood cells and MPs at baseline and after different therapy duration in AKI patients.Methods: Fifty AKI patients admitted to the First Affiliated Hospital of Harbin Medical University between March 2017 and May 2018 and forty healthy controls were enrolled in this study. Plasma samples were collected at baseline from patients were diagnosed with AKI and after receiving CRRT (Mode: CVVH; dose: 25mL/kg/h; Citrate which has relative little influence on PS exposure and MPs count was used as regional anticoagulant.) for 6hours, 12hours, 18hours, as well as from healthy controls. After being isolated, MPs were measured by flow cytometry (FCM) while procoagulant activity (PCA) of erythrocytes, platelets and MPs was assessed through coagulation time and purified coagulation complex. Lactadherin was applied as a probe and inhibitor of PS.Results: Between AKI baseline group and healthy controls, there was no obvious difference in lactadherin+ MPs, exposure on platelets and erythrocytes (Table 1,2), coagulation time and protein production (Fig. 1). While with therapy time prolonged to 6h, 12h and 18h, lactadherin+ MPs and blood cells showed a significant increase compared with baseline group (Table 1,2). Additionally, coagulation time shortened significantly (Fig. 1A) and purified coagulation complex increased visibly in parallel with total therapy time (Fig. 1B-D). Based on previous analysis, we used lactadherin to suppress PS exposure at time point 18h. Compared with the baseline, lactadherin+ MPs count declined by 75.5% (P < 0.001), coagulation time for MPs, platelets and erythrocytes was shortened by 74.6%, 77.2% and 71.9% (all p < 0.01) and procoagulant enzyme complexes were also reduced by 73.8, 78.7% and 72.9% (all p < 0.01). (Figure. 1)Conclusions: Our results first reveal that upon AKI patients on CRRT, a procoagulant role of PS-driven PCA and an immediate time-dependent elevation in PS+ MPs and blood cells is observed. What's more, suppression of PS obviously inhibits the PCA. Accordingly, PS shows potential to be a new predictor and new strategies focusing on blockade of PS may provide a novel way of anticoagulants in CRRT process. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Tyrosine Kinase Inhibitor Pazopanib Inhibits Platelet Procoagulant Activity in Renal Cell Carcinoma Patients.

Pazopanib is an angiostatic tyrosine kinase inhibitor (TKI) presently used for cancer treatment, particularly in patients with renal cell carcinoma (RCC). This treatment can be accompanied by mild bleeding as an adverse effect. Given the role of protein tyrosine kinases in platelet activation processes, we investigated whether and how pazopanib can affect platelet functions in purified systems and during treatment of advanced RCC patients. In isolated platelets from healthy volunteers, pazopanib dose-dependently reduced collagen-induced integrin activation and secretion, as well as platelet aggregation. Pazopanib addition diminished glycoprotein (GP) VI-dependent tyrosine phosphorylation of multiple platelet proteins, including the tyrosine kinase Syk. Furthermore, pazopanib inhibited GPVI-induced Ca2+ elevation, resulting in reduced exposure of the procoagulant phospholipid phosphatidylserine (PS). Upon perfusion of control blood over a collagen surface, pazopanib inhibited thrombus size as well as PS exposure. Blood samples from 10 RCC patients were also analyzed before and after 14 days of pazopanib treatment as monotherapy. This treatment caused an overall lowering in platelet count, with 3 out of 10 patients experiencing mild bleeding. Platelets isolated from pazopanib-treated patients showed a significant lowering of PS exposure upon activation. In addition, platelet procoagulant activity was inhibited in thrombi formed under flow conditions. Control experiments indicated that higher pazopanib concentrations were required to inhibit GPVI-mediated PS exposure in the presence of plasma. Together, these results indicated that pazopanib suppresses GPVI-induced platelet activation responses in a way partly antagonized by the presence of plasma. In treated cancer patients, pazopanib effects were confined to a reduction in GPVI-dependent PS exposure. Together with the reduced platelet count, this may explain the mild bleeding tendency observed in pazopanib-treated patients.

Loss of the mitochondrial kinase PINK1 does not alter platelet function

PTEN-induced putative kinase (PINK) 1 is regarded as a master regulator of cellular mitophagy such that loss of function mutations contribute to early onset Parkinson’s disease, through aberrant mitochondrial control and function. Mitochondrial function is key to platelet procoagulant activity, controlling the haemostatic response to vessel injury, but can also predispose blood vessels to thrombotic complications. Here, we sought to determine the role of PINK1 in platelet mitochondrial health and function using PINK1 knockout (KO) mice. The data largely show an absence of such a role. Haematological analysis of blood counts from KO mice was comparable to wild type. Quantification of mitochondrial mass by citrate synthase activity assay or expression of mitochondrial markers were comparable, suggesting normal mitophagy in KO platelets. Analysis of mitochondrial permeability transition pore opening, changes in mitochondrial membrane potential and calcium signalling to platelet activation were unaffected by loss of PINK1, whereas subtle enhancements of activation-induced reactive oxygen species were detected. Platelet aggregation, integrin activation, α- and dense granule secretion and phosphatidylserine exposure were unaltered in KO platelets while mouse tail bleeding responses were similar to wild type. Together these results demonstrate that PINK1 does not regulate basal platelet mitophagy and is dispensable for platelet function.

Multifaceted effects of arachidonic acid and interaction with cyclic nucleotides in human platelets

IntroductionArachidonic acid induced aggregation is a generally accepted test for aspirin resistance. However, doubts have been raised that arachidonic acid stimulated aggregation can be regarded as reliable testing for aspirin resistance. Arachidonic acid, in addition to platelet activation, can induce phosphatidylserine translocation on the outer surface of platelet membrane which could be mediated by apoptosis pathways or transformation of platelets to the procoagulant state. Materials and methodsWe explored effects of arachidonic acid over a vast range of concentrations and a wide range of read-outs for human platelet activation, procoagulant activity, and platelet viability. Additionally we tested whether cAMP- or cGMP-dependent protein kinase activation can inhibit procoagulant activity or platelet viability. ResultsArachidonic acid-induced washed platelet activation was detected at low micromolar concentrations during the first 2 min of stimulation. After longer incubation and/or at higher concentrations arachidonic acid triggered platelet procoagulant activity and reduced platelet viability. At the same time, arachidonic acid stimulated adenylate cyclase mediated protein phosphorylation which correlated with reduced platelet activation. Moreover, additional stimulation of cAMP- or cGMP-dependent protein kinase inhibited only platelet activation, but did not prevent pro-coagulant activity and platelet death. ConclusionsWhile arachidonic acid induces platelet activation at low concentrations and during short incubation time, higher concentrations and lasting incubation evokes adenylate cyclase activation and subsequent protein phosphorylation corresponding to reduced platelet activation, but also enhanced pro-coagulant activity and reduced viability. Our observations provide further proof for the complex fine tuning of platelet responses in a time and agonist concentration dependent manner.

P6559APAC, a dual antiplatelet and anticoagulant, attenuates platelet procoagulant activity and localizes to extravascular matrix during vascular injury

P6559APAC, a dual antiplatelet and anticoagulant, attenuates platelet procoagulant activity and localizes to extravascular matrix during vascular injury