Significant Differences In Platelet Function Research Articles

Comparison of platelet proteomic profiles between children and adults reveals origins of functional differences.

Platelets are blood cells responsible for the prevention of blood loss upon vessel wall disruption. It has been demonstrated that platelet functioning differs significantly between adult and pediatric donors. This study aimed to identify potential differences between the protein composition of platelets of pediatric, adolescent, and adult donors. Platelet functional testing was conducted with live cell flow cytometry. Using a straightforward approach to platelet washing based on the sequential platelets centrifugation-resuspension, we were able to obtain stable and robust proteomics results, which corresponded to previously published data. We have identified that pediatric donors' platelets have increased amounts of proteins, responsible for mitochondrial activity, proteasome activity, and vesicle transport. Flow cytometry analysis of platelet intracellular signaling and functional responses revealed that platelets of the pediatric donors have diminished granule secretion and increased quiescent platelet calcium concentration and decreased calcium mobilization in response to ADP. We could explain the observed changes in calcium responses by the increased mitochondria protein content, and the changes in granule secretion could be explained by the differences in vesicle transport protein content. Therefore, we can conclude that the age-dependence of platelet functional responses originates from the difference in platelet protein content. Platelets of infants are known to functionally differ from the platelet of adult donors, although the longevity and persistivity of these differences are debatable. Pediatric donor platelets have enhanced amounts of mitochondrial, proteasomal, and vesicle transport proteins. Platelets of the pediatric donors had increased cytosolic calcium in the resting state, what is explained by the increased numbers of mitochondrial proteins. Infants had decreased platelet granule release, which resolved upon adolescence. Thus, platelets of the infants should be assessed differently from adult platelets. Differences in platelet proteomic contents persisted in adolescent groups, yet, no significant differences in platelet function were observed.

Association of platelet function with depression and its treatment with sertraline in patients with chronic kidney disease: analysis of a randomized trial

BackgroundMajor Depressive Disorder (MDD) can lead to adverse cardiovascular outcomes in patients with chronic kidney disease (CKD). Although one of the proposed mechanisms is heightened platelet activation, effects of MDD and its treatment with a selective serotonin reuptake inhibitor (SSRI) on platelet function in patients with CKD remain unclear.MethodsIn a pre-specified analysis, changes from baseline to 12 weeks in whole blood platelet aggregation (WBPA) and plasma levels of E-selectin and P-selectin on treatment with sertraline vs. placebo were investigated in 175 patients with CKD (estimated glomerular filtration rate [eGFR] < 60 ml/min/1.73m2) and MDD (MDD+/CKD+) in a randomized, double-blind trial. Correlations between severity of depressive symptoms and platelet function were also analyzed. In order to investigate whether differences in platelet function were due to presence of CKD or MDD, we compared a subgroup of 49 MDD+/CKD+ patients with eGFR < 30 ml/min/1.73m2 to 43 non-depressed CKD controls (28 CKD with eGFR < 30 ml/min/1.73m2 [MDD−/CKD+] and 15 individuals with eGFR ≥90 ml/min/1.73m2 [MDD−/CKD-].ResultsIn MDD+/CKD+ individuals, there were no significant correlations between severity of depressive symptoms and platelet function, and no significant changes in platelet function after 12 weeks of treatment with sertraline vs. placebo. There were no significant differences in platelet function among MDD+/CKD+ patients and controls without MDD except in WBPA to 10 μM ADP (P = 0.03). WBPA to ADP was lower in the MDD−/CKD- group (8.0 Ω [5.0 Ω, 11.0 Ω]) as compared to the MDD−/CKD+ group (12.5 Ω [8.0 Ω, 14.5 Ω]), P = 0.01, and the MDD+/CKD+ group (11.0 Ω [8.0 Ω, 15.0 Ω]), P < 0.01.ConclusionsHeightened ADP-induced platelet aggregability was observed in CKD patients compared to controls with normal kidney function, regardless of presence of comorbid MDD, and treatment with sertraline did not affect platelet function. These findings suggest that increased platelet activation may not be a major contributory underlying mechanism by which depression may lead to worse cardiovascular outcomes in patients with CKD. Future studies should include positive MDD controls without CKD to confirm our findings.Trial registrationClinicalTrials.gov identifier numbers: CAST Study: NCT00946998 (Recruitment Status: Completed. First Posted: July 27, 2009. Results First Posted: January 30, 2018). WiCKDonASA Study: NCT01768637 (Recruitment Status: Completed. First Posted: January 15, 2013. Results First Posted: April 19, 2019).

Modulation of Platelet Functions Assessment during Menstruation and Ovulatory Phases.

During menstruation, endometrial hemostasis is achieved by platelet aggregation, fibrin deposition, and thrombus formation that interact with local endocrine and immunological factors which cause termination of menstrual bleeding. Interactions between steroidal sex hormones and platelet functions are not well understood. The aim of this study was to evaluate the effect of platelet function during the menstrual cycle and luteal phase in women of reproductive age. The cross-sectional study on women of reproductive age included 44 healthy women. Platelet function was assessed by PFA-100TM analyzer with collagen/epinephrine and collagen/ADP cartridges during the menstrual cycle and luteal phase. There were no significant differences in platelet function between menstruation and ovulatory phase. Platelet activity in Arab collagen/epinephrine cartridge increased during menstruation compared to non-Arab ethnic subjects and no significant differences in platelet function were found when using collagen/ADP cartridge. This study suggested modulation in platelet functions during menstruation and luteal phase in women of reproductive age. Further studies, including a large number of subjects, platelet genetic and progesterone factors change in platelet clotting associated to menstrual cycle should be conducted.

The norpurpureine alkaloid from Annona purpurea inhibits human platelet activation in vitro

BackgroundThe leaves of Annona purpurea have yielded several alkaloids with anti-aggregation activities against rabbit platelets. This is promising in the search for agents that might act against platelets and reduce the incidence of cardiovascular diseases. Since significant differences in platelet function have been reported between human and animal platelets, a study focusing on the effect of A. purpurea extracts against human platelet activation is necessary.MethodsThe compounds in an A. purpurea ethanolic extract underwent bio-guided fractionation and were used for in vitro human platelet aggregation assays to isolate the compounds with anti-platelet activity. The bioactive compounds were identified by spectroscopic analysis. Additional platelet studies were performed to characterize their action as inhibitors of human platelet activation.ResultsThe benzylisoquinoline alkaloid norpurpureine was identified as the major anti-platelet compound. The IC50 for norpurpureine was 80 μM against platelets when stimulated with adenosine 5′-diphosphate (ADP), collagen and thrombin. It was pharmacologically effective from 20 to 220 μM. Norpurpureine (220 μM) exhibited its in vitro effectiveness in samples from 30 healthy human donors who did not take any drugs during the 2 weeks prior to the collection. Norpurpureine also gradually inhibited granule secretion and adhesion of activated platelets to immobilized fibrinogen. At the intra-platelet level, norpurpureine prevented agonist-stimulated calcium mobilization and cAMP reduction. Structure–activity relationship analysis indicates that the lack of a methyl group at the nitrogen seems to be key in the ability of the compound to interact with its molecular target.ConclusionNorpurpureine displays a promising in vitro pharmacological profile as an inhibitor of human platelet activation. Its molecular target could be a common effector between Ca2+ and cAMP signaling, such as the PLC-PKC-Ca2+ pathway and PDEs. This needs further evaluation at the protein isoform level.

αIIb(R990W), a Constitutive Activating Mutation of Integrin αIIbβ3, Knock-in Mice Show Macrothrombocytopenia with Impairment of Platelet Function

Background Integrin αIIbβ3 plays an essential role in platelet aggregation and quantitative or qualitative defects in αIIbβ3 lead to severe bleeding diathesis, which is known as Glanzmann thrombasthenia (GT). Although platelet counts and morphology are usually normal in GT patients, we and others found that several mutations around transmembrane regions of αIIb or β3, which cause constitutive activation of αIIbβ3, are associated with macrothrombocytopenia. Among them, αIIb(R995W) was found in four unrelated Japanese families, suggesting that the mutation may be the most prevalent in Japanese regarding αIIbβ3-associated macrothrombocytopenia (Kunishima, Kashiwagi, et al. Blood. 2011;117:5479-84). We also found reduction of surface expression of αIIbβ3 on platelets in patients with αIIbβ3-associated macrothrombocytopenia, suggesting that activating mutations may affect platelet function via not only activating state of αIIbβ3 but also its expression (Kashiwagi, et al. Mol Genet Genomic Med. 2013;1:77-86). In this study, we generated αIIb(R990W), which is corresponding to human αIIb(R995W), knock-in (KI) mice and analyzed the effects of constitutive activation of αIIbβ3 on platelet numbers, morphology and function.Methods: αIIb(R990W) mutation was introduced to C57BL/6-derived ES cells by homologous recombination and KI mice were generated. Reticulated platelets were detected by flow cytometry after thiazole orange staining. Platelet life span was assessed by flow cytometry after injection with NHS-biotin. Platelet function was assessed by light transmission aggregometry and platelet adhesion/aggregation on collagen-coated glass under shear stress in vitro. Platelet function was also assessed by tail bleeding time and thrombus formation induced by FeCl3injury on mesenteric artery in vivo.Results: Hetero and Homo KI mice were born in accordance with Mendel’s laws and showed no obvious bleeding tendency. Platelet counts were decreased in KI mice [WT: 1.22±0.11(x106/μl), Hetero: 1.10±0.14*, Homo: 0.91±0.11* (*p<0.05 compared with WT)] and platelet size assessed by forward side scatter of flow cytometry showed mild increase of platelet size in KI mice [WT: 26.3±0.60 (arbitrary unit), Hetero: 27.7±0.56*, Homo: 32.5±0.98*(*p<0.05 compared with WT)]. Absolute reticulated platelet numbers of Homo were decreased compared with WT [WT: 102±21.9 (x103/μl), Hetero: 84.0±23.7, Homo: 66.6±12.3*(*p<0.05 compared with WT)]. There was no difference in serum thrombopoietin levels among Homo, Hetero and WT mice. Platelet recovery after thrombocytopenia caused by anti-GPIb antibodies was mildly impaired on 4 days after injection in KI mice. These results suggest that platelet production may be mildly impaired in KI mice. Platelet life span was also slightly but significantly shortened in KI mice compared with WT, which may also contribute to thrombocytopenia in KI mice. Surface expression of αIIbβ3 in Hetero and Homo platelets was decreased to 74.0±3.4 and 3.3±0.6% of WT, whereas that on megakaryocytes in Hetero and Homo was 81.0±15.2% and 62.3±8.9% of WT, respectively. Bleeding time of Homo mice was markedly prolonged [WT: 2.44±2.08 min (n=12), Hetero: 3.14±2.62 min (n=12), Homo: >10min in 8 of 10 mice]. Thrombus formation on collagen-coated coverslip under flow conditions as well as platelet aggregation induced by collagen and PAR4-TRAP was markedly impaired in Homo platelets. Homo mice also showed markedly impaired thrombus formation in FeCl3-induced thrombus model. There were no significant differences in platelet function between Hetero and WT.Conclusion: The αIIbβ3 activating mutation, αIIb(R990W), leads to macrothrombocytopenia in mice. Homo KI mice also show marked reduction of surface αIIbβ3 expression in platelet, leading to severe impairment of platelet aggregation and thrombus formation like GT. DisclosuresNo relevant conflicts of interest to declare.

Abstract 576: ABO Blood Group and Platelet Aggregation

Background: ABO blood groups have been linked to coronary thrombosis, with non-O blood groups at higher risk of thrombosis. ABO blood group antigens are expressed on platelet glycoproteins, including the IIb fibrinogen receptor, and may affect platelet function via glycan modifications. We hypothesized that non-O blood groups would demonstrate higher levels of platelet aggregation compared to type O blood group. Methods: Healthy, non-smoking subjects were recruited to a protocol investigating glycan modification of platelets. Ex vivo platelet aggregation was assessed by light transmission aggregometry of platelet-rich plasma in dose-response to collagen (0.04-0.2 mg/mL) and thrombin receptor activating peptide (TRAP) (1-5 μmol). Maximal platelet aggregation data, aggregation rate and lag time were compared at each concentration across blood groups A, B, AB and O by ANOVA testing. Results: A total of 91 subjects were recruited (ABO types; 32 A, 16 B, 8 AB, 35 O). The average age of the subjects was 31 years, 58% were Caucasian and 61% were female. There were no significant differences in age, race and gender between ABO blood groups. For collagen concentrations ranging from 0.04-0.2 mg/mL, there was no significant difference in mean platelet aggregation, rate of aggregation and lag time between blood groups. For TRAP concentrations between 1-5 μmol, there was also no significant difference in mean platelet aggregation and rate of aggregation between blood groups. For TRAP at 2 μmol and 3 μmol, blood group AB demonstrated a longer lag time compared to blood groups A, B, and O (TRAP 2 μmol; 13 sec type AB vs 5.21 sec type A, 4.18 sec type B, 3.57 sec type O, TRAP 3 μmol; 9.12 sec type AB vs 3.34 sec type A, 4.31 sec type B, 2.65 sec type O) but these secondary analyses were of marginal significance after consideration of multiple testing. Conclusion: These data for ex vivo platelet aggregation dose-response to collagen and TRAP do not support significant differences in platelet function by ABO blood type. Further studies with larger sample sizes and of specific platelet glycoprotein functions are required to address potential subtle influences of ABO glycans on platelet reactivity.

Women with Red Hair Report a Slightly Increased Rate of Bruising but Have Normal Coagulation Tests

There is an anecdotal impression that redheads experience more perioperative bleeding complications than do people with other hair colors. We, therefore, tested the hypothesis that perceived problems with hemostasis could be detected with commonly used coagulation tests. We studied healthy female Caucasian volunteers, 18 to 40 yr of age, comparable in terms of height, weight, and age, with natural bright red (n = 25) or black or dark brown (n = 26) hair. Volunteers were questioned about their bleeding history and the following tests were performed: complete blood count, prothrombin time/international normalized ratio, activated partial thromboplastin time, platelet function analysis, and platelet aggregation using standard turbidimetric methodology. Agonists for aggregation were adenosine diphosphate, arachidonic acid, collagen, epinephrine, and two concentrations of ristocetin. The red-haired volunteers reported significantly more bruising, but there were no significant differences between the red-haired and dark-haired groups in hemoglobin concentration, platelet numbers, prothrombin time/international normalized ratio, or activated partial thromboplastin time. Furthermore, no significant differences in platelet function, as measured by platelet function analysis or platelet aggregometry, were observed. We conclude that if redheads have hemostasis abnormalities, they are subtle.

Reduction of bleeding after heart operations through the prophylactic use of epsilon-aminocaproic acid

Excessive postoperative bleeding after heart operations continues to be a source of morbidity. This prospective double-blind study evaluated epsilon-aminocaproic acid as an agent to reduce postoperative bleeding and investigated its mode of action. One hundred three patients were randomly assigned to receive either 30 gm epsilon-aminocaproic acid (51 patients) or an equivalent volume of placebo (52 patients). In a subset of these patients (14 epsilon-aminocaproic acid, 12 placebo), tests of platelet function and fibrinolysis were performed. Results: By multivariate analysis, three factors were associated with decreased blood loss in the first 24 hours after operation: epsilon-aminocaproic acid versus placebo (647 ml versus 839 ml, p = 0.004), surgeon 1 versus all other surgeons (582 ml versus 978 ml, p = 0.002), and no intraaortic balloon versus intraaortic balloon pump use (664 ml versus 1410 ml, p = 0.02). No significant differences in platelet function could be demonstrated between the two groups. Inhibited fibrinolysis, as reflected by less depression of the euglobulin clot lysis and no rise in d-dimer levels, was significant in the epsilon-aminocaproic acid group compared with the placebo group. Conclusion: The intraoperative use of epsilon-aminocaproic acid reduces postoperative cardiac surgical bleeding. (J T HORAC C ARDIOVASC S URG 1996;112:1098-107)

Thrombocytopenia-adapted in vitro bleeding test assesses platelet function in thrombocytopenic patients.

Platelet counts do not always reflect the true bleeding risk in chronically thrombocytopenic patients, and the posttransfusion platelet increments do not necessarily demonstrate that therapeutic efficacy. There are no easy and reliable tests yet permitting the determination of platelet function in thrombocytopenic patients. The in-vitro bleeding test (IVBT) with the Thrombostat 4000 proved to be a very sensitive and specific test for the detection of platelet disorders. In order to become suitable for the investigation of thrombocytopenic blood with platelet count between 5 x 10(9)/L and 50 x 10(9)/L, special modifications were necessary. We report on the evaluation of two thrombocytopenia-adapted modifications (TP-IVBT 150/120), first with blood of healthy donors made thrombocytopenic (three experiments with six blood samples each of different platelet concentrations and identical hematocrit) and then in a clinical study on 77 thrombocytopenic patients (69 with bone marrow hypoplasia, eight with autoimmune thrombocytopenia) receiving 267 platelet transfusions. The patients were followed over 15 days on average (1-67 days) by daily examinations (total 1,285 observation days). Most TP-IVBT measurements were carried out in triplicate, using the modification with the 120-microns filter (TP-IVBT 120) because it proved to be superior to the other modification. Additionally, cell counts, hematocrit, body temperature, platelet volume, platelet distribution width, expression of CD 36, 41a, 42b on platelets, Simplate bleeding time, and detailed analysis of bleeding signs were performed for the calculation of a bleeding score. There was a close correlation between TP-IVBT and platelet counts with thrombocytopenic normal blood (r2 = 0.81-0.94). This indicated the suitability of this test modification to examine platelet function in thrombocytopenic patients. The clinical study showed that the TP-IVBT helped at least to determine the platelet-related bleeding risk in thrombocytopenic patients. It allowed differentiation between hypoplastic and autoimmune thrombocytopenia in most cases. In addition, significant differences in platelet function of various diseases and of different bone marrow regeneration could be demonstrated. The TP-IVBT is well-suited for the control of platelet transfusion efficacy and may replace the in-vivo bleeding time in most cases. On the other hand, the test still shows too much of a variation and involves too much labor and cost for routine application.

A contribution to the indications for platelet transfusion and determination of its therapeutic efficacy

The platelet count does not really reflect the true bleeding risk in chronically thrombocytopenic patients. Recently, we reported on two modifications of an in vitro bleeding test (IVBT) which appeared to be suitable for the evaluation of primary non-vascular hemostatis in thrombocytopenic and anemic patients (platelets 10–50,000/μL, hct 16–30 L/L). We report on the clinical study conducted with the IVBT modification which proved to be superior. Fifty thrombocytopenic patients, 42 with bone marrow hypoplasia and 8 with autoimmune thrombocytopenia, were followed up for a total of 686 days and received 161 platelet transfusions (mainly from cell separator). The IVBT was carried out with the Thrombostat 4000 R in triplicate using 120 μm filters and evaluating the occlusion time (OT). Additionally, cell count, hematocrit, body temperature, platelet volume, platelet distribution width, Simplate R time and a detailed analysis of bleeding signs for the calculation of a bleeding score were performed. The IVBT modification used allowed the determination of platelet-related bleeding risk in thrombocytopenic patients. With the IVBT, significant differences in platelet function in different patients could be demonstrated which primarily reflected the underlying disease. In addition, bone marrow regeneration correlated with platelet function. From the findings of this study, the authors formulate criteria for the indication of platelet transfusion which includes platelet function as well as the platelet count. Increased individual bleeding risk factors have to be considered too. But before generalization these criteria have to be verified by a controlled prospective study. In addition, the IVBT was well suited to prove the efficacy of platelet transfusions. On the other hand, the test still involves too much labor and cost for routine application. The next generation of equipment and improved disposables should solve these problems.

Platelet Function and Serum High Density Lipoproteins

Two groups of healthy male subjects one with low (mean 1.03 mmol/l), and the other with normal (mean 1.80 mmol/l), concentrations of high density lipoprotein (HDL) cholesterol in plasma were investigated. A series of in vitro platelet function tests were performed in order to investigate a possible connection between low HDL concentration, which has been defined as an independent risk factor for the occurrence of CHD, and disturbances in platelet function. No significant differences in platelet function were observed between the two groups. A positive correlation was found between platelet factor 3 activity both in platelet rich and platelet poor plasma and the plasma concentration of HDL.