Hemoperfusion leads to impairment in hemostasis and coagulation process in patients with acute pesticide intoxication

Abstract Hemoperfusion (HP) is one of the important treatment modalities in extracorporeal therapy for patients with acute intoxication. Its use has declined during the past 20 years despite its efficacy, because of its side effects, especially an increased risk of bleeding. Mechanisms of hemostasis impairment have not been clearly elucidated and studies demonstrating the mechanism are lacking. It is not clear which step of the hemostatic process is impaired during HP, and whether it leads to an increased risk of bleeding. We performed both in vivo and in vitro studies to elucidate the mechanism of impairment in the hemostatic process. In patients with acute pesticide intoxication who underwent HP, the platelet count decreased rapidly during the first 30 minutes from 242.4 ± 57.7 × 103/μL to 184.8 ± 49.6 × 103/μL, then gradually decreased even lower to 145.4 ± 61.2 × 103/μL over time (p < 0.001). As markers of platelet activation, platelet distribution width increased continuously during HP from 41.98 ± 9.28% to 47.69 ± 11.18% (p < 0.05), however, mean platelet volume did not show significant change. In scanning electron microscopy, activated platelets adhered to modified charcoal were observed, and delayed closure time after HP in PFA-100 test suggested platelet dysfunction occurred during HP. To confirm these conflicting results, changes of glycoprotein expression on the platelet surface were evaluated when platelets were exposed to modified charcoal in vitro. Platelet expression of CD61, fibrinogen receptor, significantly decreased from 95.2 ± 0.9% to 73.9 ± 1.6%, while those expressing CD42b, von Willebrand factor receptor, did not show significant change. However, platelet expression of CD49b, collagen receptor, significantly increased from 24.6 ± 0.7% to 51.9 ± 2.3%. Thrombin-antithrombin complex, a marker for thrombin generation, appeared to decrease, however, it was not statistically significant. Fibrin degradation products and d-dimers, markers for fibrinolysis, increased significantly during HP. Taken together, our data suggests that hemoperfusion leads to impairment of platelet aggregation with incomplete platelet activation, which was associated with reduced thrombin generation, accompanied by increased fibrinolysis.

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Thrombin Generation Measured Ex Vivo Following Microvasculor Injury Is Increased in SLE Patients with Antiphospholipid-protein Antibodies

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657710 ◽

1997 ◽

Vol 78 (04) ◽

pp. 1173-1177 ◽

Cited By ~ 15

Author(s):

Jacek Musiał ◽

Jakub Swadźba ◽

Miłosz Jankowski ◽

Marek Grzywacz ◽

Stanisława Bazan-Socha ◽

...

Keyword(s):

Lupus Erythematosus ◽

Thrombin Generation ◽

Ex Vivo ◽

Skin Incision ◽

Anticardiolipin Antibodies ◽

Small Blood Vessel ◽

Anionic Phospholipids ◽

Increased Risk ◽

High Concentrations

SummaryAntiphospholipid-protein antibodies (APA) include lupus-type anticoagulant (LA) and antibodies recognizing complexes of anionic phospholipids (e.g. cardiolipin) and proteins (e.g. prothrombin and (β2-glycoprotein I). The presence of APA is associated with an increased risk of both arterial and venous thrombosis. However, the pathogenic mechanism leading to thrombosis in patients with APA remains unclear. We studied 32 patients with systemic lupus erythematosus (SLE) who were divided into two groups depending on the presence (n = 19) or absence (n = 13) of APA. Healthy volunteers (n = 12) matched by age and sex served as controls. In all subjects LA and IgG class anticardiolipin antibodies (ACA) were determined. Thrombin generation was monitored ex vivo measuring fibrinopeptide A (FPA) and prothrombin fragment F1 + 2 (F1 + 2) in blood emerging from a skin microvasculature injury, collected at 30 second intervals. In subjects with antiphospholipid antibodies mean FPA and F1 + 2 concentrations were signiF1cantly higher at most blood sampling times than in controls. In some SLE patients with APA the process of thrombin generation was clearly disturbed and very high concentrations of F1brinopeptide A were detected already in the F1rst samples collected. Two minutes after skin incision SLE patients without APA produced slightly more FPA, but not F1 + 2, as compared to healthy subjects. Mathematical model applied to analyze the thrombin generation kinetics revealed that APA patients generated signiF1cantly greater amounts of thrombin than healthy controls (p = 0.02 for either marker). In contrast, in the same patients generation of thrombin in recalciF1ed plasma in vitro was delayed pointing to the role of endothelium in the phenomenon studied. In summary, these data show for the F1rst time that in SLE patients with antiphospholipid-protein antibodies thrombin generation after small blood vessel injury is markedly increased. Enhanced thrombin generation might explain thrombotic tendency observed in these patients.

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Impaired Platelet Function in Sept8-Deficient Mice In Vitro

Thrombosis and Haemostasis ◽

10.1055/s-0040-1718733 ◽

2020 ◽

Author(s):

Kerstin Jurk ◽

Katharina Neubauer ◽

Victoria Petermann ◽

Elena Kumm ◽

Barbara Zieger

Keyword(s):

Platelet Function ◽

Thrombin Generation ◽

Human Platelets ◽

Platelet Surface ◽

Integrin Αiibβ3 ◽

Glycoprotein Vi ◽

Fibrinogen Receptor ◽

Static Conditions ◽

The Impact

AbstractSeptins (Septs) are a widely expressed protein family of 13 mammalian members, recognized as a unique component of the cytoskeleton. In human platelets, we previously described that SEPT4 and SEPT8 are localized surrounding α-granules and move to the platelet surface after activation, indicating a possible role in platelet physiology. In this study, we investigated the impact of Sept8 on platelet function in vitro using Sept8-deficient mouse platelets. Deletion of Sept8 in mouse platelets caused a pronounced defect in activation of the fibrinogen receptor integrin αIIbβ3, α-granule exocytosis, and aggregation, especially in response to the glycoprotein VI agonist convulxin. In contrast, δ-granule and lysosome exocytosis of Sept8-deficient platelets was comparable to wild-type platelets. Sept8-deficient platelet binding to immobilized fibrinogen under static conditions was diminished and spreading delayed. The procoagulant activity of Sept8-deficient platelets was reduced in response to convulxin as determined by lactadherin binding. Also thrombin generation was decreased relative to controls. Thus, Sept8 is required for efficient integrin αIIbβ3 activation, α-granule release, platelet aggregation, and contributes to platelet-dependent thrombin generation. These results revealed Sept8 as a modulator of distinct platelet functions involved in primary and secondary hemostatic processes.

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Sulfated poly-amido-saccharides (sulPASs) are anticoagulants in vitro and in vivo

Chemical Science ◽

10.1039/d1sc02302k ◽

2021 ◽

Vol 12 (38) ◽

pp. 12719-12725

Author(s):

Maria Varghese ◽

Rae S. Rokosh ◽

Carolyn A. Haller ◽

Stacy L. Chin ◽

Jiaxuan Chen ◽

...

Keyword(s):

Murine Model ◽

Protamine Sulfate ◽

Clotting Time ◽

Risk Of Bleeding ◽

Increased Risk

Heparin mimicking sulfated poly-amido-saccharides (sulPASs) are anticoagulants resistant to heparanases and reversed by protamine sulfate. In an in vivo murine model, sulPASs extend clotting time without the increased risk of bleeding.

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Partially desulfated heparin modulates the interaction between anti-protamine/heparin antibodies and platelets

Thrombosis and Haemostasis ◽

10.1160/th15-07-0539 ◽

2016 ◽

Vol 115 (02) ◽

pp. 324-332 ◽

Cited By ~ 4

Author(s):

Rabie Jouni ◽

Heike Zöllner ◽

Ahmad Khadour ◽

Jan Wesche ◽

Anne Grotevendt ◽

...

Keyword(s):

Platelet Activation ◽

Platelet Factor ◽

Standard Drug ◽

Platelet Surface ◽

Minimal Impact ◽

Platelet Survival ◽

Heparin Complexes ◽

The Impact

SummaryProtamine (PRT) is the standard drug to neutralise heparin. PRT/heparin complexes induce an immune response similar to that observed in heparin-induced thrombocytopenia (HIT). Partially desulfated heparin (ODSH) was shown to interfere with anti-platelet factor 4/heparin antibodies (Abs), which are responsible for HIT. In this study, we analyse the impact of ODSH on the interaction between anti-PRT/heparin Abs and platelets. The ability of ODSH to prevent anti-PRT/heparin Ab-induced platelet destruction in vivo was investigated using the NOD/ SCID mouse model. ODSH improved platelet survival in the presence of PRT, heparin and anti-PRT/heparin Abs (median platelet survival after 300 minutes (min) with 20 μg/ml ODSH: 75 %, range 70–81 % vs without ODSH: 49%, range 44–59%, p=0.006). Furthermore, when ODSH was applied 60 min after Ab injection platelet survival was improved (median platelet survival after 300 min with ODSH: 83 %, range 77–93 % vs without ODSH: 59 %, range 29–61 %, p=0.02). In in vitro experiments ODSH inhibited platelet activation at concentrations > 16 μg/mL (p< 0.001), as well as PRT/heparin complex binding to platelets (mean fluorescence intensity [MFI] without ODSH: 85 ± 14 vs with ODSH: 15 ± 0.6, p=0.013). ODSH also displaced pre-bound complexes from the platelet surface (MFI without ODSH: 324 ± 43 vs with 32 μg/ml ODSH: 53 ± 9, p< 0.001). While interfering with platelet activation by anti-PRT/heparin Abs, up to a concentration of 16 μg/ml, ODSH had only minimal impact on neutralisation of heparin by PRT. In conclusion, our study shows that ODSH is able to inhibit platelet activation and destruction suggesting a potential clinical use to reduce anti-PRT/heparin Ab-mediated adverse effects.

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Injection of recombinant activated factor VII can induce transient increase in circulating procoagulant microparticles

Thrombosis and Haemostasis ◽

10.1160/th03-05-0301 ◽

2004 ◽

Vol 91 (05) ◽

pp. 873-878 ◽

Cited By ~ 21

Author(s):

Bénédicte Hugel ◽

Benoit Guillet ◽

Catherine Trichet ◽

Anne Rafowicz ◽

Thierry Lambert ◽

...

Keyword(s):

Short Duration ◽

Platelet Activation ◽

Thrombin Generation ◽

Transient Increase ◽

Factor Vii ◽

Recombinant Activated Factor Vii ◽

Activated Factor Vii ◽

Therapeutic Doses

SummaryRecombinant activated factor VII (rFVIIa) is an effective haemostatic treatment in haemophiliacs with inhibitors. In vitro, FVIIa concentrations corresponding to those obtained with therapeutic doses of rFVIIa have been shown to induce normal thrombin generation and platelet activation in the absence of factors VIII or IX. To further study the in vivo haemostatic changes induced by rFVIIa, circulating procoagulant microparticles (MP) were measured in patients treated with discontinuous injections of Novoseven®. In 6 out of 15 patients, a transient peak of procoagulant MP was observed after injection, occurring 15 min to 2 h after infusion. It was composed primarily of platelet-derived MP and was of very short duration. This peak was not observed in haemophiliacs without inhibitor, who were treated with conventional replacement therapies. Our results provide further in vivo evidence that rFVIIa specifically activates platelets, either directly or as a consequence of a burst of thrombin generation that could account for its haemostatic efficacy.

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Activity Pattern Analysis Indicates Increased but Balanced Systemic Coagulation Activity in Response to Surgical Trauma

TH Open ◽

10.1055/s-0038-1673390 ◽

2018 ◽

Vol 02 (04) ◽

pp. e350-e356

Author(s):

Max Friedrich ◽

Jan Schmolders ◽

Yorck Rommelspacher ◽

Andreas Strauss ◽

Heiko Rühl ◽

...

Keyword(s):

Activity Pattern ◽

Plasma Levels ◽

Thrombin Generation ◽

Pattern Analysis ◽

Activity Level ◽

Surgical Trauma ◽

Coagulation Activity ◽

Increased Risk ◽

D Dimer

AbstractIn the nonbleeding patient, constant low-level activation of coagulation enables a quick procoagulant response upon an injury. Conversely, local activation of coagulation might influence the systemic activity level of coagulation. To characterize this interaction in more detail, activity pattern analysis was performed in patients undergoing elective surgeries. Blood samples were taken before, during, and 24 hours after surgery from 35 patients undergoing elective minor (n = 18) and major (n = 17) orthopaedic surgeries. Plasma levels of thrombin and activated protein C (APC) were measured using oligonucleotide-based enzyme capture assays, while those of prothrombin fragment 1.2, thrombin–antithrombin-complexes, and D-dimer were measured using commercially available enzyme-linked immunosorbent assays. In vitro thrombin generation kinetics were recorded using calibrated automated thrombography. Results showed that median plasma levels of up to 20 pM thrombin and of up to 12 pM APC were reached during surgery. D-dimer levels started to increase at the end of surgery and remained increased 24 hours after surgery, while all other parameters returned to baseline. Peak levels showed no significant differences between minor and major surgeries and were not influenced by the activity state at baseline. In vitro thrombin generation kinetics remained unchanged during surgery. In summary, simultaneous monitoring of the procoagulant and anticoagulant pathways of coagulation demonstrates that surgical trauma is associated with increased systemic activities of both pathways. Activity pattern analysis might be helpful to identify patients at an increased risk for thrombosis due to an imbalance between surgery-related thrombin formation and the subsequent anticoagulant response.

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Platelet Distribution Width Is Associated with P-Selectin Dependent Platelet Function: Results from the Moli-Family Cohort Study

Cells ◽

10.3390/cells10102737 ◽

2021 ◽

Vol 10 (10) ◽

pp. 2737

Author(s):

Benedetta Izzi ◽

Alessandro Gialluisi ◽

Francesco Gianfagna ◽

Sabatino Orlandi ◽

Amalia De Curtis ◽

...

Keyword(s):

Platelet Activation ◽

Platelet Function ◽

Ex Vivo ◽

Venous Blood ◽

Random Effect ◽

Platelet Distribution Width ◽

Mixed Effect ◽

Distribution Width ◽

Inflammatory Conditions

Defined as an index of platelet size heterogeneity, the platelet distribution width (PDW) is still a poorly characterized marker of platelet function in (sub)clinical disease. We presently validated PDW as a marker of P-selectin dependent platelet activation in the Moli-family cohort. Platelet-bound P-selectin and platelet/leukocyte mixed aggregates were measured by flow cytometry in freshly collected venous blood, both before and after in vitro platelet activation, and coagulation time was assessed in unstimulated and LPS- or TNFα-stimulated whole blood. Closure Times (CT) were measured in a Platelet Function Analyzer (PFA)-100. Multivariable linear mixed effect regression models (with age, sex and platelet count as fixed and family structure as random effect) revealed PDW to be negatively associated with platelet P-selectin, platelet/leukocyte aggregates and von Willebrand factor (VWF), and positively with PFA-100 CT, and LPS- and TNF-α-stimulated coagulation times. With the exception of VWF, all relationships were sex-independent. In contrast, no association was found between mean platelet volume (MPV) and these variables. PDW seems a simple, useful marker of ex vivo and in vitro P-selectin dependent platelet activation. Investigations of larger cohorts will define the usefulness of PDW as a risk predictor of thrombo-inflammatory conditions where activated platelets play a contributing role.

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Abstract 456: Antiphospholipid Antibodies Promote Platelet Activation and Thrombosis by Inhibition of Platelet eNOS

Circulation ◽

10.1161/circ.116.suppl_16.ii_76-c ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

Craig Morrell ◽

AnneMarie Swaim ◽

Tanika Martin ◽

Guillermina Girardi ◽

Jane E Salmon ◽

...

Keyword(s):

Platelet Activation ◽

Antiphospholipid Antibodies ◽

Thrombus Formation ◽

Ldl Receptor ◽

Wild Type ◽

Human Igg ◽

Increased Risk ◽

Receptor Family

The antiphospholipid syndrome (APS) is an autoimmune systemic disorder characterized by the persistent presence of antiphospholipid antibodies (aPL Ab) and increased risk of thrombosis, coronary artery disease and myocardial infarction. Although platelets are known direct targets of aPL Ab action, the molecular basis of aPL Ab actions on platelets remains unclear. Platelet endothelial NO synthase (eNOS) is a key regulator of platelet function, with NO causing blunted activation. We therefore determined whether aPL Ab modulate platelet eNOS. Normal human IgG (NH IgG) and human IgG containing polyclonal aPL Ab were obtained from healthy individuals and APS patients, respectively, and purified using protein G-Sepharose chromatography. Using both human and mouse platelets, we found that aPL Ab increased agonist-induced platelet activation whereas NH IgG did not. In contrast to the enhanced activation by aPL Ab in platelets from wild-type mice, aPL Ab had no effect on platelets isolated from eNOS null mice. Pre-treatment of platelets with aPL Ab also inhibited insulin-mediated eNOS stimulation as evidenced by diminished cGMP production and DAF2 fluorescence. Receptor associated protein (RAP), an antagonist of ligand binding to members of the LDL receptor family, blocked aPL Ab-induced increases in platelet activation. RAP also prevented aPL Ab-mediated antagonism of platelet eNOS, indicating that aPL Ab signal through the platelet ApoER2â ϵ™ (LRP8) to attenuate eNOS activity. Furthermore, using intravital microscopy of the mouse mesenteric circulation, we demonstrated that platelets from wild-type mice treated with aPL Ab have increased rolling on a stimulated endothelium and a decreased time to thrombus formation in vivo versus platelets treated with NH IgG. In contrast, aPL Ab did not alter the in vivo function of platelets from eNOS null mice. These cumulative in vitro and in vivo findings demonstrate that aPL Ab antagonism of platelet eNOS through LDL receptor family member binding underlies aPL Ab-mediated thrombosis.

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Imer Blocks Phosphorylation of the β3 Integrin, Decreasing Platelet Activation Responses and Protecting Mice From Arterial Thrombosis

Blood ◽

10.1182/blood.v118.21.189.189 ◽

2011 ◽

Vol 118 (21) ◽

pp. 189-189

Author(s):

Brian R. Branchford ◽

Susan Sather ◽

Gary Brodsky ◽

Tara C White-Adams ◽

Deborah DeRyckere ◽

...

Keyword(s):

Carotid Artery ◽

Platelet Activation ◽

Ex Vivo ◽

Human Platelets ◽

Fibrillar Collagen ◽

Carotid Artery Injury ◽

Platelet Surface ◽

Β3 Integrin ◽

Platelet Spreading

Abstract Abstract 189 Background: Growth Arrest Specific gene 6 (Gas6) signaling through platelet-surface Tyro3/Axl/Mer (TAM) receptors leads to platelet activation and thrombus stabilization. This occurs via activation of phosphatidylinositol-3-kinase (PI3K) and Akt, stimulating tyrosine phosphorylation of the β3 integrin. This process amplifies outside-in signaling via αIIbβ3, which is necessary for stable aggregate formation. iMer is a truncated form of the extracellular domain of the Mer receptor tyrosine kinase, produced by alternative splicing, that inhibits Gas6/TAM signaling, likely by acting as a decoy receptor for Gas6. Objectives: We hypothesized that inhibiting the Gas6/TAM pathway with a novel Gas6-sequestering protein would decrease platelet activation responses. We therefore evaluated iMer's inhibition of Gas6 signaling in human and murine platelets in vitro and in vivo. Methods: We measured the inhibitory effect of iMer on platelet activation using laboratory evaluations of platelet function and a murine carotid artery thrombosis model. In vitro studies included aggregometry, adhesion to collagen in a flow chamber, and platelet spreading. These platelet activation responses were tested in human platelets in the presence or absence of the inhibitor and also in wild type (WT) and Gas6/TAM −/− murine platelets. A ferric-chloride model of carotid artery injury was used to compare susceptibility to thrombosis between littermate C57BL/6 mice treated with either iMer or vehicle. Platelet aggregation data was evaluated by the Wilcoxon Signed Rank Test, and times to occlusion following carotid artery injury were compared using the Mann-Whitney Rank Sum Test. Results/Discussion: Western blot analysis demonstrated decreased β3 integrin phosphorylation in iMer-treated human platelets after addition of human Gas6 when compared to controls, consistent with decreased Mer signaling in the presence of iMer. iMer-treated human platelets exhibited significant decreases in ADP- and collagen-induced platelet aggregation. ADP-stimulated samples treated ex vivo with iMer showed an aggregation mean of 74% (SD= +/− 3%), compared to 86% aggregation (+/− 3%) in controls (p=0.016). Collagen-stimulated samples treated ex vivo with iMer exhibited a mean of 70% aggregation (+/− 8%), compared to 88% aggregation (+/−2%) in controls (p=0.004). Electron micrographs of adhered human platelets revealed that iMer delayed, but did not permanently abrogate, platelet spreading on fibrillar collagen (100 μg/mL). Flow cytometric analysis of human platelets showed reduced expression of platelet-surface activation markers (P-selectin and PAC-1) despite stimulation with fibrillar collagen (1 μg/mL). Microfluidic flow assay demonstrated that adhesion of untreated human platelets to collagen at a wall shear rate of 100s−1 resulted in 21.3% (SD=+/− 8%) mean surface area coverage, while ex vivo iMer-treated samples showed only 1.1% (+/− 0.9%) coverage. These results are consistent with those of WT mice compared to that of Gas6/TAM −/−mice in preliminary studies using the same system. Following ferric chloride injury to the carotid artery, 71% of vehicle-treated control mice (n=7) had initial occlusions that remained stable, and only 14% remained patent. In contrast, only 25% of the iMer-treated mice (n=8) formed initial occlusions that remained stable, while 50% remained patent. The iMer treated mice also had a significant decrease (p=0.02) in the duration of first occlusion time (i.e. length of time the initial occlusion lasted), suggesting decreased thrombus stability. Conclusions: iMer is a novel inhibitor of the Gas6/TAM pathway that decreases platelet activation responses and protects mice from arterial thrombosis by decreasing phosphorylation of β3 integrin, which has been shown to be necessary for thrombus stabilization. This compound may, therefore, have translational applications as a novel anti-platelet agent. Disclosures: No relevant conflicts of interest to declare.

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Platelets, vascular disease, and diabetes mellitus

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y94-045 ◽

1994 ◽

Vol 72 (3) ◽

pp. 295-303 ◽

Cited By ~ 34

Author(s):

Peter D. Winocour

Keyword(s):

Diabetes Mellitus ◽

Vascular Disease ◽

Platelet Activity ◽

Platelet Surface ◽

Clinical Complications ◽

Fibrinogen Binding ◽

Increased Risk ◽

Adhesive Proteins

Diabetes is associated with increased risk for atherosclerosis and its thromboembolic complications. Theories about mechanisms of atherosclerosis in diabetes are similar to those in the nondiabetic population. Platelets contribute to atherosclerosis through effects on vessels by materials released from the platelets, which interact with injured or altered vessels. In diabetes, platelets could contribute to enhanced atherosclerosis through hypersensitivity to agonists at sites of vessel injury and increased release of materials from adherent platelets. Diabetic platelets are hypersensitive to agonists in vitro, and alterations in a number of mechanisms involved in platelet activation occur in these platelets, which could contribute to the hypersensitivity. These alterations include increased presence of glycoprotein receptors for agonists and adhesive proteins on the platelet surface, increased fibrinogen binding, decreased membrane fluidity, enhanced arachidonate pathway activation with increased thromboxane A2 formation, and increased phosphoinositide turnover leading to increased inositol trisphosphate production, Ca2+ mobilization, and protein phosphorylation. There is some evidence for increased platelet activity in vivo in diabetes, but it is unclear whether this reflects platelet hypersensitivity or increased platelet turnover on already diseased vessels. Studies in diabetic animals indicate greater interaction of platelets with injured vessels and incorporation into experimentally induced thrombi, but it is unclear if this reflects changes in platelets or other factors. These changes could be contributing to the enhanced atherosclerosis and its clinical complications in diabetic patients.Key words: platelets, vascular disease, diabetes mellitus.

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