The Analysis of Mutual Relationship Between Hemostasis and Inflammatory Processes in Patients with Chronic Liver Diseases

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1015-1015
Author(s):  
Nora E. El-Bassiouni ◽  
Rania M. Benyamin ◽  
Samia H. Risk ◽  
Manal Y. Zahran ◽  
Ola M. Mahmoud ◽  
...  
Keyword(s):  
Liver Cirrhosis ◽  
Platelet Count ◽  
Platelet Activation ◽  
Healthy Subjects ◽  
Blood Platelet ◽  
Surface Expression ◽  
Strongly Correlated ◽  
Blood Monocytes ◽  
Monocyte Activation ◽  
Platelet Surface

Abstract Introduction: Monocytes play a crucial role in the pathogenesis of inflammation and fibrosis in chronic liver diseases (CLD). Platelet, by connecting hemostasis and inflammatory processes, also participate in the pathogenesis of CLD. We aimed to assess the expression of platelet monocyte aggregates and their relationship to both monocyte and platelet activation in patients with hepatitis C virus (HCV) induced CLD in relation to the degree of hepatic insufficiency and haemostatic imbalance. Methods: Sixty patients with HCV induced CLD were categorized after Child Pugh criteria into 4 groups: Child A, B, C and C during acute attack of haematemesis, 15 patients each, 15 healthy subjects were also included as normal controls. Immunophenotype characterization for identification of platelet monocyte aggregates (CD41/CD45) on activated blood monocytes (CD11b and CD14) and activated blood platelet populations (CD61and CD62P) was carried out by flowcytometric analysis. The circulating levels of monocyte activation marker (MCP-1) and platelet activation markers (PF4 and sP-selectin) were also determined using immunological assays. Results: A marked reduction in platelet count concomitant with a decrease in the CD61 platelets surface expression were noticed in different groups of patients with HCV (p <0.01) compared to healthy subjects, implicating that the platelet count drops in correlation with advancement of liver cirrhosis. Data demonstrated a significant increase (p <0.01) in the surface expression of each of CD11b, CD14 and CD41/45 on peripheral blood monocytes revealing increased monocyte activation in patients with HCV compared to controls, especially Child C groups. These findings revealed that the increased expression of CD11b on blood monocytes matches the advancement of liver failure and that the CD14 is up regulated in viral hepatitis and correlate with liver injury. MCP-1 concentration did not correlate with monocyte count in peripheral blood denoting that MCP-1 level did not reflect the inflammatory activity in patients with liver cirrhosis, as it was lower in blood serum than in healthy subjects. Moreover, a significant progressive increase (p <0.01) in the expression of CD62P and CD41/CD45 on platelets and in the circulating levels of sP-selectin and PF4 was also detected in different groups of patients with HCV compared to controls. These findings revealed increased platelet activation with the rise in population of CD62P phenotype in patients with liver cirrhosis despite thrombocytopenia intensification. Our results also showed that the CD62P platelet surface expression was inversely correlated with platelet count and was strongly correlated with sP-selectin levels in Child B and C groups. Likewise, PF4 levels were strongly correlated with CD62P platelet surface expression and inversely correlated with platelet count in both groups revealing that the more developed thrombocytopenia, the stronger the platelet activation in liver cirrhosis. Our data also demonstrate that CD41/45 surface expression of blood platelet monocyte aggregate on platelets and monocytes are inversely correlated with platelet count and strongly correlated with each of monocyte activation (CD11b and CD14) and platelet activation (sP-selectin, CD62P and PF4) markers in patients with Child B and C showing that as cirrhosis develops and platelet count diminishes platelet aggregations with monocytes increase. Conclusions: Monocytes and platelets activation in patients with HCV increased in stepwise fashion in conjunction with worsening severity of liver disease as assessed by Child Pugh staging. The interplay of several factors such as endotoxaemia, lipopolysaccharides, oxidative stress and proinflammatory cytokines may be implicated in this respect. Also, the permanent flow of leukocytes and platelets through inflamed liver tissues and their long exposure to the active proinflammatory factors may lead to the activation of these cells. In turn, the progressive increase in monocytes and platelets activation with the simultaneous formation of monocyte-platelet aggregates noticed in patients with HCV may contribute to progressive liver injury in these patients. We, therefore, suggest that the blockage of platelet and monocyte activation may diminish thrombocytopenia and liver cirrhosis intensification in patients with chronic hepatitis C virus. Disclosures No relevant conflicts of interest to declare.

Post-DDAVP Thrombocytopenia in Type 2B von Willebrand Disease Is not Associated with Platelet Consumption: Failure to Demonstrate Glycocalicin Increase or Platelet Activation

1999 ◽  
Vol 81 (02) ◽  
pp. 224-228 ◽  
Author(s):  
A. Steffan ◽  
E. Pontara ◽  
A. Zucchetto ◽  
C. Rossi ◽  
L. De Marco ◽  
...  
Keyword(s):  
Platelet Count ◽  
Platelet Activation ◽  
Surface Expression ◽  
Von Willebrand Disease ◽  
Platelet Surface ◽  
Platelet Counts ◽  
Normal Platelet ◽  
Thrombotic Complications ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryThrombocytopenia is frequently reported in type 2B von Willebrand disease (vWD), and thought to be related to the abnormally high affinity of 2B von Willebrand factor (vWF) for platelet GPIb-IX. To gain an insight into the nature of this thrombocytopenia, we measured plasma glycocalicin (GC) levels (as a marker of platelet turnover), and platelet surface expression of the alpha granule protein P-selectin (as a marker of platelet activation) in 9 patients with type 2B vWD before, and in 4 patients also following the infusion of 1-desamino-8-d-arginine vasopressin (DDAVP). Three patients presented a persistent decrease of platelet counts in the resting condition. GC levels were within the normal range, regardless of the platelet counts, in all but one patient who presented, on the other hand, a normal platelet count. Moreover, platelets expressed normal amounts of P-selectin on their surface, regardless of platelet counts. These findings suggest that the thrombocytopenia observed in type 2B vWD is not due to platelet activation and subsequent consumption in circulation.Despite a significant, albeit transient, decrease in platelet count, DDAVP did not induce an increase in plasma GC levels, nor enhance P-selectin expression. These observations indicate that the acute post-DDAVP thrombocytopenia in type 2B vWD is not related to platelet activation and consumption. We advance that the post-DDAVP 2B vWF is hemostatically more active, and able to induce agglutination but not aggregation of circulating platelets. This would explain both the prompt recovery of basal platelet counts after the post-DDAVP decrease, and the lack of reported thrombotic complications in this disorder.Therefore, even though 2B vWF is characterized by an enhanced affinity for the platelet surface, its binding to platelet GPIb-IX in the soluble phase is not able to induce true platelet aggregation; vWF thus appears to be mainly an adhesive protein, rather than an aggregating agent.

Distinct serotypes of streptococcal M proteins mediate fibrinogen-dependent platelet activation and pro-inflammatory effects

2021 ◽  
Author(s):  
Frida Palm ◽  
Sounak Chowdhury ◽  
Sara Wettemark ◽  
Johan Malmström ◽  
Lotta Happonen ◽  
...  
Keyword(s):  
Platelet Activation ◽  
M Protein ◽  
Multiparameter Flow Cytometry ◽  
Invasive Infection ◽  
Monocyte Activation ◽  
Platelet Surface ◽  
Sequence Variations ◽  
M Proteins ◽  
Inflammatory State ◽  
Life Threatening

Sepsis is a life-threatening complication of infection that is characterised by a dysregulated inflammatory state and disturbed hemostasis. Platelets are the main regulators of hemostasis, and they also respond to inflammation. The human pathogen Streptococcus pyogenes can cause local infection that may progress to sepsis. There are more than 200 different serotypes of S. pyogenes defined according to sequence variations in the M protein. The M1 serotype is among ten serotypes that are predominant in invasive infection. M1 protein can be released from the surface and has previously been shown to generate platelet, neutrophil and monocyte activation. The platelet dependent pro-inflammatory effects of other serotypes of M protein associated with invasive infection (M3, M5, M28, M49 and M89) is now investigated using a combination of multiparameter flow cytometry, ELISA, aggregometry and quantitative mass spectrometry. We demonstrate that only M1-, M3- and M5 protein serotypes can bind fibrinogen in plasma and mediate fibrinogen and IgG dependent platelet activation and aggregation, release of granule proteins, upregulation of CD62P to the platelet surface, and complex formation with neutrophils and monocytes. Neutrophil and monocyte activation, determined as upregulation of surface CD11b, is also mediated by M1-, M3- and M5 protein serotypes, while M28-, M49- or M89 proteins failed to mediate activation of platelets or leukocytes. Collectively, our findings reveal novel aspects of the immunomodulatory role of fibrinogen acquisition and platelet activation during streptococcal infections.

scholarly journals von Willebrand factor bound to glycoprotein Ib is cleared from the platelet surface after platelet activation by thrombin

Blood ◽  
1992 ◽  
Vol 79 (8) ◽  
pp. 2011-2021 ◽  
Author(s):  
P Hourdille ◽  
HR Gralnick ◽  
E Heilmann ◽  
A Derlon ◽  
AM Ferrer ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Platelet Activation ◽  
Von Willebrand Factor ◽  
Platelet Rich Plasma ◽  
Surface Expression ◽  
Von Willebrand Disease ◽  
Platelet Surface ◽  
Immunogold Staining ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract We recently reported that after activation of human platelets by thrombin, glycoprotein (GP) Ib-IX complexes are translocated to the surface-connected canalicular system (SCCS) (Blood 76:1503, 1990). As GPIb is a major receptor for von Willebrand factor (vWF) in platelet adhesion, we have now examined the consequences of thrombin activation on the organization of vWF bound to GPIb on the platelet surface. Studies were performed using monoclonal or polyclonal antibodies in either immunogold staining and electron microscopy (Au-EM) or in flow cytometry. When unstirred platelet-rich plasma was incubated with ristocetin, bound vWF was located by Au-EM as discrete masses regularly distributed over the cell surface. Platelets from a patient with Glanzmann's thrombasthenia, lacking GPIIb-IIIa complexes, gave a similar pattern, confirming that this represented binding to GPIb. That ristocetin was not precipitating vWF before their binding to the platelets was shown by the detection of similar masses on the surface of platelets of a patient with type IIB von Willebrand disease. Experiments were continued using washed normal platelets incubated in Tyrode-EDTA, the purpose of the EDTA being to limit the surface expression of endogenous vWF after platelet stimulation. Under these conditions, platelets were treated with ristocetin for 5 minutes at 37 degrees C in the presence of increasing amounts of purified vWF. This was followed by incubation with thrombin (0.5 U/mL) for periods of up to 10 minutes. Flow cytometry showed a time-dependent loss in the surface expression of vWF bound to GPIb and these changes were confirmed by Au-EM. In particular, immunogold staining performed on ultrathin sections showed that the bulk of the vWF was being cleared to internal membrane systems. Surface clearance of vWF during thrombin- induced platelet activation is a potential mechanism for regulating platelet adhesivity.

Influence of platelet count on the expression of platelet collagen receptor glycoprotein VI (GPVI) in patients with acute coronary syndrome

2009 ◽  
Vol 101 (05) ◽  
pp. 911-915 ◽  
Author(s):  
Boris Bigalke ◽  
Konstantinos Stellos ◽  
Dimitrios Stakos ◽  
Thomas Joos ◽  
Oliver Pötz ◽  
...  
Keyword(s):  
Acute Coronary Syndrome ◽  
Platelet Count ◽  
Thrombus Formation ◽  
Surface Expression ◽  
Platelet Surface ◽  
Glycoprotein Vi ◽  
Coronary Syndrome ◽  
Symptomatic Coronary Artery Disease ◽  
Artery Disease ◽  
Collagen Receptor

SummaryPlatelets play a key role in the development of an acute coronary syndrome (ACS) and contribute to cardiovascular events. Platelet collagen receptor glycoprotein VI (GPVI) contributes significantly to platelet adhesion and thrombus formation in ACS. We consecutively investigated both the platelet count and the platelet surface expression of GPVI in 843 patients with a symptomatic coronary artery disease verified by coronary angiography. Four hundred fourteen patients presented with stable angina pectoris and 429 patients with ACS. Platelet surface expression of GPVI and CD62P was determined by flow cytometry and platelet count with a coulter counter, plasmatic soluble GPVI was measured by ELISA. Platelet GPVI expression in patients with ACS was compared to platelet count. Patients with ACS showed significantly elevated GPVI expression levels in the first and second quartiles of platelet count compared to patients with higher platelet count [mean fluorescence intensity (MFI) ± standard deviation): 1st vs. 4th: 20.44 ± 6.1 vs. 18.62 ± 3.7; p=0.012; 2ndvs.3rd:21.2±8.5vs.18.76±3.7;P=0.03; 2ndvs.4th: 21.2±8.5vs.18.62±3.7;P=0.004], which was paralleled in trend for the CD62P expression [MFI: 1st vs. 4th: 11.2 ± 6.8 vs. 12.3 ± 9; p=0.057; 2nd vs. 3rd: 16.3 ± 16 vs.12.7 ± 5.3; p=0.138; 2nd vs. 4th: 16.3 ± 16 vs.11 ± 4.4; p=0.043]. In a subgroup of 48 patients with ACS, determination of soluble GPVI showed similar results [plasma GPVI (ng/ml): 1stvs.4th: 1.6 ± 0.6 vs. 1.2 ± 0.4; p=0.046; 1st vs. 3rd: 1.6 ± 0.6 vs. 1.1 ± 0.5; p=0.038; 2nd vs. 3rd: 1.9 ± 0.8 vs. 1.1 ± 0.5; p=0.04; 2nd vs. 4th: 1.9 ± 0.8 vs. 1.2 ± 0.4; p=0.056]. Thus, a lower platelet count comes along with a higher GPVI surface expression and plasma concentration in patients with ACS, which potentially reflects increased activation and enhanced recruitment of platelets to the site of vascular injury.

Analysis of Platelet Receptor Expression in ITP,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3289-3289
Author(s):  
Jianlin Qiao ◽  
Huy Tran ◽  
Fi-Tjen Mu ◽  
Robert K Andrews ◽  
Elizabeth E Gardiner
Keyword(s):  
Platelet Count ◽  
Conflicts Of Interest ◽  
Surface Expression ◽  
Experimental Models ◽  
Receptor Expression ◽  
Response To Treatment ◽  
Platelet Surface ◽  
Healthy Donors ◽  
Platelet Receptor ◽  
Von Willebrand

Abstract Abstract 3289 In this study we assess platelet receptor expression and shedding in patients with immune thrombocytopenia (ITP) before and during treatment. The aim is to evaluate the potential value of quantitative measurement of platelet receptors for diagnosis and/or monitoring treatment in thrombocytopenia due to immune or other causes. The platelet-specific collagen receptor, glycoprotein (GP)VI, is associated with the Fc receptor γ-chain (FcRγ). GPVI/FcRγ is coassociated on platelet surface with the GPIb-IX-V complex; GPIbα of GPIb-IX-V binds von Willebrand factor and other ligands. Our previous studies showed engagement of platelet FcγRIIa by antiplatelet antibodies induced ectodomain shedding of GPVI, generating soluble ectodomain (sGPVI) in plasma. However, apart from one individual with an anti-GPVI antibody, whether anti-platelet antibodies associated with ITP affect GPVI/GPIb expression/shedding has not been addressed. In this study we used flow cytometry and a sGPVI ELISA to assess 1) whether patients with ITP had dysregulated expression/shedding of GPVI or GPIbα, and 2) whether platelet receptor expression changes prior to recovery of platelet count in individuals undergoing treatment for ITP. In 9 ITP patients (mean age=48.6, range 29–79; 6 female) with platelet count 61±9 × 109/L (range, 33–122 × 109/L), GPVI surface expression (GeoMean±SE, 137±17) was lower than healthy controls (274±26; n=17; platelet count 247±13), and sGPVI in patient plasma was significantly higher (39±4 ng/mL) compared to 17 healthy donors (19±3 ng/mL) (P=0.0006). In longitudinal samples analysed at weekly intervals during 2-month treatment with steroids, decreased GPVI surface expression and increased sGPVI in plasma remained essentially unchanged as the platelet count normalized, consistent with persistent anti-platelet antibody. However, while levels of intact platelet GPIbα were significantly reduced in ITP compared to healthy donors (P=0.0053), they approached healthy levels within 1 week of treatment, preceding improvement in platelet count or other measures. GPIbα expression/cleavage has been previously implicated in platelet clearance in experimental models, and our analysis suggests the proteolytic status of human GPIbα may be a novel early marker for evaluating response to treatment in ITP. Disclosures: No relevant conflicts of interest to declare.

Association Of Platelet Function Markers, Independent Of Platelet Count, With Bleeding Score In Patients With Immune Thrombocytopenia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3534-3534
Author(s):  
Andrew L. Frelinger ◽  
Anja J Gerrits ◽  
Michelle A. Berny-Lang ◽  
Travis Brown ◽  
Sabrina L. Carmichael ◽  
...  
Keyword(s):  
Platelet Count ◽  
Platelet Activation ◽  
Platelet Function ◽  
Research Funding ◽  
Immune Thrombocytopenia ◽  
Univariate Analysis ◽  
Blood Draw ◽  
Platelet Surface ◽  
Platelet Counts ◽  
Bleeding Score

Abstract Background Immune thrombocytopenia (ITP) patients with similarly low platelet counts differ in their tendency to bleed. Aim To determine if differences in platelet function in ITP patients with similarly low platelet counts partly account for the variation in bleeding tendency. Methods The relationship between bleeding scores and platelet function markers was investigated in a single center cross-sectional study of pediatric patients with ITP. Following informed consent, blood was collected from ITP patients and bleeding was graded using the Buchanan and Adix Score (J Pediatr 2002) at routine clinic visits or while admitted to the hospital. Bleeding scores were obtained by one of three hematologists blinded to platelet function results, and investigators performing platelet function tests were blinded to clinical results. Platelet function was assessed by whole blood flow cytometric measurement of unstimulated, ADP- or TRAP-stimulated platelet surface activated GPIIb-IIIa (as measured by PAC1 binding), P-selectin, and GPIb and by unstimulated, convulxin-, or ADP plus TRAP-stimulated platelet surface phosphatidylserine expression (as determined by annexin V binding). Platelet count, immature platelet fraction (IPF) and mean platelet volume (MPV) were determined by a Sysmex XE-2100, and platelet forward angle light scatter (FSC) was measured by flow cytometry. Results Platelet function and bleeding scores were evaluated in 34 consecutive consenting pediatric ITP patients (16 female, 18 male, age 9.7 ± 5.7 years [mean ± SD]). ITP was newly diagnosed (< 3 months) in 10 patients, persistent (3 -- 12 months) in 7 patients, and chronic (>12 months) in 17 patients. Platelet count at the time of the blood draw was 47 ± 55 x 109/L. The median bleeding score on day of blood draw was 1 (range 0 to 4). By univariate analysis, higher IPF, and lower platelet count were significantly associated with a higher bleeding score (odds ratio [OR] >1, p<0.05) but MPV was not. Multiple measures of platelet function were associated with bleeding scores by univariate analysis: higher levels of platelet FSC (a measure affected by multiple variables including size) surface GPIb on unstimulated, ADP- or TRAP-stimulated platelets, surface P-selectin on unstimulated platelets, and platelet FSC were associated with increased odds for higher bleeding scores (ORs each >1, p<0.05), while higher ADP- and TRAP-stimulated platelet surface activated GPIIb-IIIa and P-selectin were associated with reduced odds of higher bleeding scores (ORs each <1, p<0.05). After adjustment for platelet count, higher levels of platelet surface P-selectin on unstimulated platelets, GPIb on TRAP-stimulated platelets, and FSC remained significantly associated with increased odds for higher bleeding scores (Figure), but IPF did not. Similarly, after adjustment for platelet count, higher TRAP-stimulated percentage of P-selectin and activated GPIIb-IIIa positive platelets remained significantly associated with reduced odds of higher bleeding scores (Figure). These findings were independent of recent ITP-related treatment. Conclusions In this study of pediatric ITP patients, we identified selected platelet function markers which, independent of platelet count, are associated with increased (platelet FSC, platelet surface P-selectin on unstimulated platelets, and GPIb on TRAP-stimulated platelets) or decreased (TRAP-stimulated percent P-selectin and GPIIb-IIIa positive platelets) odds of high bleeding scores. Possible hypotheses to explain these associations are as follows: 1) Increased P-selectin on unstimulated platelets demonstrates in vivo platelet activation, possibly as a consequence of the recent bleeding. 2) Because platelet activation results in a reduction in platelet surface GPIb and increases in platelet surface activated GPIIb-IIIa and P-selectin, the ORs associated with all of these markers could be explained by reduced ability of platelets in patients with higher bleeding scores to respond to agonists. 3) While platelet FSC is partly related to size, the finding that MPV and IPF, adjusted for platelet count, were not associated with bleeding score suggests that factors other than size account for the association of platelet FSC with higher bleeding scores. Further study is required to validate these findings and determine if differences in platelet function are associated with future risk for bleeding. Disclosures: Off Label Use: Eltrombopag was given to WAS/XLT patients for treatment of thrombocytopenia. Neufeld:Shire: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Apopharma: Consultancy. Michelson:Sysmex: Honoraria.

scholarly journals The Changes of Blood Platelet Reactivity in the Presence of Crude Extracts Isolated from Leaves and Twigs of Elaeagnus Rhamnoides (L.) A. Nelson Measuring in Whole Blood

2021 ◽  
Author(s):  
Bartosz Skalski ◽  
Joanna Rywaniak ◽  
Jerzy Żuchowski ◽  
Anna Stochmal ◽  
Beata Olas
Keyword(s):  
Phenolic Compounds ◽  
Platelet Activation ◽  
Whole Blood ◽  
Leaf Extract ◽  
Platelet Reactivity ◽  
Blood Platelets ◽  
Blood Platelet ◽  
Surface Expression ◽  
Sea Buckthorn ◽  
Crude Extracts

Abstract Uncontrolled blood platelet activation is an important risk factor of cardiovascular disease (CVDs). Various studies on phenolic compounds indicate that they have a protective effect on the cardiovascular system through different mechanisms, including the reduction of blood platelet activation. One of the plants that is particularly rich in phenolic compounds is sea buckthorn (Elaeagnus rhamnoides (L.) A. Nelson). The aim of the present study in vitro was to determine the anti-platelet properties of crude extracts isolated from leaves and twigs of E. rhamnoides (L.) A. Nelson in whole blood using flow cytometric and total thrombus-formation analysis system (T-TAS). The aim of our study was also analyze of blood platelet proteoms in the presence of different sea buckthorn extracts. A significant new finding is a decrease surface expression of P-selectin on blood platelets stimulated by 10 µM ADP and 10 µg/mL collagen, and a decrease surface expression of GPIIb/IIIa active complex on non-activated platelets and platelets stimulated by 10 µM ADP and 10 µg/mL collagen in the presence of sea buckthorn leaf extract (especially at the concentration 50 µg/mL). The twig extract also displayed antiplatelet potential. However, this activity was higher in the leaf extract than in the twig extract in whole blood. In addition, our present findings clearly demonstrate that investigated plant extracts have anticoagulant properties (measured by T-TAS). Therefore, the two tested extracts may be promising candidates for the natural anti-platelet and anticoagulant supplements.

A Study of Platelet Activation during Human Cardiopulmonary Bypass and the Effect of S-Nitrosoglutathione

1997 ◽  
Vol 78 (06) ◽  
pp. 1516-1519 ◽  
Author(s):  
Edward J Langford ◽  
Andrew Parfitt ◽  
Adam J de Beider ◽  
Michael T Marrinan ◽  
John F Martin
Keyword(s):  
Nitric Oxide ◽  
Flow Cytometry ◽  
Cardiac Surgery ◽  
Cardiopulmonary Bypass ◽  
Platelet Activation ◽  
Surface Expression ◽  
Platelet Inhibition ◽  
Nitric Oxide Donor ◽  
Platelet Dysfunction ◽  
Platelet Surface

SummaryCardiac surgery is complicated by the occurrence of post-operative bleeding due to platelet dysfunction. This is largely caused by platelet activation and consumption during cardiopulmonary bypass. Patients undergoing cardiac surgery requiring cardiopulmonary bypass were studied to determine whether early platelet changes due to bypass could be inhibited using the platelet-selective nitric oxide donor S-nitroso-glutathione (GSNO). Flow cytometry was used to measure platelet surface expression of P-selectin (an α-granule protein) and glycoproteins (GP) IIb/IIIa and Ib (mediators of aggregation and adhesion) before and 5 and 10 min after commencing cardiopulmonary bypass, in 6 controls and 6 patients receiving GSNO 50 μg/min. Platelet P-selectin expression increased during bypass both in controls and patients receiving GSNO. Glycoproteins IIb/IIIa and Ib fell during bypass in control and GSNO-treated patients. There was no difference between control and GSNO-treated groups. Thus no significant platelet inhibition by S-nitrosoglutathione was demonstrated under these conditions.

Human Platelets Contain and Release an Active ADAMTS-13-Like Metalloprotease.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3527-3527
Author(s):  
Li Liu ◽  
Huiwan Choi ◽  
Bernardo Aubrey ◽  
Angila Bergeron ◽  
Leticia Nolasco ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Platelet Activation ◽  
Fluid Shear Stress ◽  
Vascular Endothelial Cells ◽  
Severe Infection ◽  
Surface Expression ◽  
Human Platelets ◽  
Platelet Surface ◽  
The Difference ◽  
Adhesion Ligand

Abstract The adhesion ligand von Willebrand factor (VWF) is synthesized and stored in vascular endothelial cells and megakaryocytes/platelets. These cells release VWF in response to similar stimulations. However, a longstanding observation stated that, unlike endothelial cells, platelets do not release the ultra-large form (UL) of VWF upon activation. The lack of ULVWF release may be attributed to the difference in multimerization process in the storage granules or the intrinsic proteolysis of the hyperreactive ULVWF. To examine these possibilities, we analyzed the multimer compositions of VWF stored in and released from platelets. We found that ULVWF was detected by immunobloting of washed platelet lysates in the presence, but not in the absence of 5 mM EDTA. Similarly, when stimulated with 5 μM of thrombin receptor-activating peptide (TRAP), platelets released only VWF that was similar in sizes to plasma VWF, suggesting that platelet ULVWF may have been cleaved before or during its release. In support of this hypothesis, we found that platelets expressed ADAMTS-13-like molecule by western blot of platelet lysates and flow cytometry using two different ADAMTS-13 antibodies. The surface expression of ADAMTS-13-like molecule increased significantly upon platelet activation by TRAP and ADP. Consistent with these observations, the Triton-X-100 lysates of washed platelets and supernatant of washed platelets activated by TRAP contained VWF-cleaving activity, as demonstrated by the cleavage of ULVWF strings formed on histamine-stimulated endothelial cells under fluid shear stress. Our finding is consistent with a previous report, but further showed that this ADAMTS-13-like molecule exists not only in cytoplasma, but also on the surface of platelets. More importantly, the ADAMTS-13-like molecule increased its expression on platelet surface and was released upon platelet activation. The ADAMTS-13-like molecule either in platelets or released upon activation was active in cleaving ULVWF. This intrinsic mechanism for ULVWF proteolysis may be physiologically important. It may prevent the sudden release of hyper-reactive ULVWF from platelets and serve as the second pool of ADAMTS-13 to encounter the increase in ULVWF release from endothelial cells during conditions such as severe infection. The role of the platelet membrane-bound ADAMTS-13 in hemostasis remains to be further determined.

In Vivo Effects of Eltrombopag on Human Platelet Function.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1301-1301 ◽  
Author(s):  
Bethan Psaila ◽  
James B. Bussel ◽  
Matthew D. Linden ◽  
You Fu Li ◽  
Marc R. Barnard ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Blood Flow ◽  
Platelet Count ◽  
Platelet Activation ◽  
Platelet Function ◽  
Whole Blood ◽  
Adult Patients ◽  
Platelet Surface ◽  
Integrin Αiibβ3

Abstract Eltrombopag, an orally-administered small-molecule agonist of the thrombopoietin receptor (c-Mpl), is under investigation as a treatment for immune thrombocytopenic purpura (ITP). Studies have indicated that eltrombopag does not ‘prime’ platelets for activation in vitro, and eltrombopag administration to healthy volunteers does not increase platelet surface P-selectin or activated integrin αIIbβ3 (Jenkins J. Blood 2007). However, the effects of eltrombopag on platelet function in thrombocytopenic patients in vivo, either by direct binding to c-Mpl receptors on platelets or indirectly by altering the dynamics of platelet production and causing an influx of young, large platelets, is unknown. Whole blood flow cytometry, unlike other assays of platelet function, enables measurement of platelet function in the setting of marked thrombocytopenia (Michelson. Platelets, Elsevier, 2007). As a substudy of larger treatment studies, 17 adult patients with chronic ITP received eltrombopag at a starting dose of 50 mg daily, with the possibility of an increase to 75 mg daily after 3 weeks. Blood samples were drawn pre-treatment, and after 7 and 28 days of therapy. Platelet count, mean platelet volume (MPV), and the immature platelet fraction (IPF, or reticulated platelet count) were measured using a Sysmex XE-2100. Platelet surface P-selectin and activated integrin αIIbβ3 (reported by monoclonal antibody PAC1) were measured by whole blood flow cytometry in the presence and absence of 0.5 μM ADP, 20 μM ADP, 1.5 μM thrombin receptor activating peptide (TRAP), or 20 μM TRAP. Bleeding was quantified by a comprehensive scale that allocates grades of 0 (no), 1 (minor) or 2 (marked) bleeding at 10 anatomical sites according to physical examination and/or history (Page, L.K. Br J Haematol 2007). Eleven of the 17 patients responded to eltrombopag with a rise in platelet count of >30 x 109/L. The IPF increased in responders but not non-responders (table 1). Response to eltrombopag was not predicted by pretreatment MPV or IPF. The ITP bleeding score decreased in responders over the study period in parallel with the increases in platelet count (table 1). As determined by platelet surface P-selectin and activated integrin αIIbβ3, eltrombopag did not result in platelet activation or augment ADP- or TRAP-induced platelet activation (table 2). In summary, eltrombopag increases the platelet count and reduces bleeding in responding adult patients with chronic ITP through the release of new platelets into the circulation. While bleeding is reduced in responders, eltrombopag does not result in platelet activation or augmentation of platelet activation by ADP or TRAP. This suggests that the newer platelets released by eltrombopag stimulation are not hyper-functional (or are only transiently so prior to day 7). Table 1 IPF (maximum absolute change, mean ± SEM x 109/L) Number in whom bleeding decreased Responders 57.0 ± 22.4 8/11 Non-responders 3.3 ± 1.5 1/6 Table 2 Study Day 0 7 28 MFI, mean fluorescence intensity, *P <0.05 compared with day 0 Activated αIIbβ3 MFI No agonist 11.4 11.3 9.2 Low ADP 180.3 159.4 98.4 High ADP 451.2 348.2* 251.8* Low TRAP 158.1 175.5 143.9 High TRAP 385.2 347.0 299.6 P-selectin MFI No agonist 5.5 6.6 6.2 Low ADP 48.6 43.4 38.8 High ADP 144.5 109.0 96.8 Low TRAP 113.8 114.9 107.8 High TRAP 457.3 396.3 330.9

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