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No description available.

Platelet transfusions can be necessary during treatment of patients with thrombocytopenia or impaired platelet function. Platelet function in platelet concentrates (PC) deteriorate with storage time. Studying swirling is often used to control the quality of PC’s before transfusion but the method has some disadvantages. Therefore other methods can be useful, for example impedance aggregometry (IA, Multiplate® Analyzer) to measure platelet function.      In this study the change in platelet function over time was examined in buffy coat and apheresis platelets with IA where aggregation had been induced with adenosine diphosphate (ADP) and collagen. PC’s were tested on day 1, 4 and 7 after donation. One of the main aims of this study was to evaluate if dilution to a standardized platelet concentration (800x109 platelets/L) for IA of PC’s could be used, since platelet concentration has been shown to influence aggregation. The effect of pathogen inactivation (INTERCEPT) on platelet function and the importance of fibrinogen for aggregation were also studied.      The dilution of platelet samples reduced the range of measured values and was suitable to use with collagen but not ADP. The platelet function decreased significantly over time with both agonists. There was a significant difference between pathogen inactivated and gamma irradiated PC’s with collagen activation on day 1. Fibrinogen was shown to be of importance for platelet aggregation, but other factors in plasma seem to be necessary too.      In conclusion, IA is a suitable method for following change in aggregability over time in PC’s and sample dilution reduced variation in results.

Cardiovascular disease is the most common cause of morbidity and mortality in type 2 diabetes. Short-term studies of glucagon-like peptide-1 (GLP-1)-targeted therapies suggest potential beneficial effects on cardiovascular outcomes. The mechanism behind this unexpectedly rapid effect is not known. In this study, full-length human GLP-1 receptor (GLP-1R) mRNA was cloned and sequenced from a human megakaryocyte cell line. Quantitative RT-PCR results showed that expression levels were comparable to other GLP-1R expressing tissues. Furthermore, incubation with GLP-1 and the GLP-1R agonist exenatide elicited a cAMP response in these cells. As megakaryocytes are the cellular precursors of platelets, the effect of GLP-1 and exenatide were studied in gel-filtered human platelet aggregation, where they were both shown to have an inhibitory effect on thrombin-stimulated platelet aggregation. Platelet inhibition by GLP-1 and GLP-1R agonists presents a potential mechanism for the reduced incidence of atherothrombotic events thought to be associated with GLP-1-targeted therapies.

Cardiovascular disease is the most common cause of morbidity and mortality in type 2 diabetes. Short-term studies of glucagon-like peptide-1 (GLP-1)-targeted therapies suggest potential beneficial effects on cardiovascular outcomes. The mechanism behind this unexpectedly rapid effect is not known. In this study, full-length human GLP-1 receptor (GLP-1R) mRNA was cloned and sequenced from a human megakaryocyte cell line. Quantitative RT-PCR results showed that expression levels were comparable to other GLP-1R expressing tissues. Furthermore, incubation with GLP-1 and the GLP-1R agonist exenatide elicited a cAMP response in these cells. As megakaryocytes are the cellular precursors of platelets, the effect of GLP-1 and exenatide were studied in gel-filtered human platelet aggregation, where they were both shown to have an inhibitory effect on thrombin-stimulated platelet aggregation. Platelet inhibition by GLP-1 and GLP-1R agonists presents a potential mechanism for the reduced incidence of atherothrombotic events thought to be associated with GLP-1-targeted therapies.

No / The isolation and characterisation of antiplatelet antibodies in autoimmune thrombocytopenia purpura patients (ITP) is described. Autoimmune thrombocytopenia purpura is an autoimmune disease, clinically defined by low platelet counts, normal or increased megakaryocytopoiesis and antiplatelet antibodies in serum. This study used phage display to isolate Fab antiplatelet antibodies to study the structure-function relationships of pathogenic antibodies in ITP. Out of six randomly selected colonies, four colonies reacted strongly with whole platelets in enzyme-linked immunosorbent assay (ELISA). Sequence analysis showed that all four colonies had the same DNA sequence and were the same antibody. Results of Western blotting against non-reduced human platelet lysate showed that the Fab reacted with platelet proteins with apparent molecular weights of 116, 92 and 39 kD. Furthermore, Western blotting assay against purified membrane glycoprotein IIIa demonstrated reactivity against a band with a molecular weight of 92 kD. Results from Western blotting against platelet lysate and pure platelet glycoprotein confirmed the Fab fragment recognised the platelet glycoprotein IIIa. Three out of the four phage colonies produced soluble Fab, which demonstrated reactivity against platelet autoantigens in ELISA. Further sequence analysis showed that the Fab was somatically mutated suggesting antigen drive and therefore T-cell assistance was important in the development of this antibody. One of the somatic mutations introduced an RSD amino acid sequence in the complementary determining region 1(CDR1) of the light chain, which may mimic the RGD motif of fibrinogen which binds integrin GPIIb/IIIa. This raises the possibility that somatic mutation and antigen drive have produced a pathogenic autoantibody.

Background: Platelets provide the initial haemostatic plug at sites of vascular injury. They also participate in pathological thrombosis that leads to myocardial infarction, stroke and peripheral vascular disease. The outcome of an acute myocardial infarction depends not only on the formation and stability of an occlusive thrombus, but also on the efficacy of the endogenous thrombolytic process, which allows reperfusion of the infarct related artery and prevents recurrent ischaemic episodes. Various platelet function tests are available to measure the thrombogenic potential of an individual, but the sensitivity of these tests remain questionable as most of these tests use citrated blood and measure response to a particular agonist. Endogenous thrombolysis has been a neglected entity, and its beneficial effects on cardiovascular outcomes has not been studied in depth in the past, possibly as until recently there has been no available technique to measure spontaneous thrombolytic activity in native blood. The Global Thrombosis Test (GTT) is a new point of care tests that allows us to measure time to thrombus formation (Occlusion time: OT) using native blood, avoiding the use of agonists and making the test results more physiological. The GTT also measures the time to lyse this formed thrombi without use of any lytic agents (Lysis time: LT), allowing us to measure the patient’s endogenous thrombolytic potential. Aim: Our aim in this study was to detect patients who are at risk of future thrombotic events despite dual antiplatelet therapy, either due to prothrombotic tendency or due to impaired endogenous thrombolysis, and to determine if these two parameters were correlated. Methods: GTT was used to assess the thrombotic and thrombolytic activity in healthy volunteers, and in different patient populations. 100 healthy volunteers were tested using the GTT, and a normal range was established. 300 patients admitted to hospital with a diagnosis of acute coronary syndrome (ACS) were included in the study, and tested using the GTT after they had been stabilized on dual antiplatelet therapy (Aspirin and Clopidogrel). All these patients were followed up for a year, to determine if their baseline GTT results were a predictor of recurrent cardiac events. The primary endpoint of the study was major adverse cardiovascular events (MACE), which was a composite of cardiovascular death, nonfatal myocardial infarction, or stroke at 12 months. Results: All results were analysed using statistical package SPSS version 16.0 (SPSS Inc., Chicago, Illinois). The 100 healthy volunteers were all non-smokers, and were not taking any medications. There were 55 males and 45 females, and mean age was 38±11 years (range 22-76, IQR 11). OT was normally distributed with mean OT 377.80s, and using mean ± 2SD, we derived a normal range of 185-569s (200-550s). LT demonstrated a skewed distribution with values ranging between 457 – 2934s. Using log transformation, a normal range of 592 – 1923 (600- 2000s) was established for LT. OT and LT were both prolonged in ACS patients compared to normal volunteers (p< 0.001). No association was observed between OT and risk of major adverse cardiovascular events. LT was noted to be a significant and independent predictor of MACE in a multivariate model adjusted for cardiovascular risk factors. LT ≥ 3000 s was the optimal cutoff value for predicting 6 month MACE [hazard ratio (HR): 2.48, 95% CI: 1.2-4.8, P= 0.008] and cardiovascular death [HR: 4.04, 95% CI : 1.3-12.0, P= 0.012 ] and 12 month MACE [HR:1.9, 95% CI: 1.04- 3.5,P= 0.03] and cardiovascular death [HR: 3.9,95% CI: 1.34-11.9, P= 0.013 ]. LT ≥ 3000 s was observed in 23% of ACS patients. Conclusions: Our study suggests that endogenous thrombolytic activity based on lysis of platelet rich thrombi can be assessed by the point of care GTT assay, which can help in identification of ACS patients at high risk of future cardiac events. Prolongation of OT may be explained by the antiplatelet effects of Aspirin and Clopidogrel, as both these drugs prolong time to thrombus formation and hence increase OT. Further large studies are required to study factors which can reduce thrombogenic potential, and improve endogenous thrombolytic activity, which can be monitored using the GTT to improve cardiovascular outcomes.

Introduction: Hemostasis is a complex system in the body that maintains blood flow and prevents bleeding. Patients with platelet disorders are at the risk of mucocutaneous bleedings and at Clinical chemistry in Linköping platelet function is measured with flow cytometry. The platelet response to various agonists is measured with fluorescently labeled platelet antibodies. The aim of this study was to evaluate the stability of the frozen reagents used for platelet function testing. Further the durability of platelet antibodies after freezing and freeze-drying was tested.   Method: Platelet antibodies were prepared for freezing/freeze-drying in buffer and were analyzed at three different occasions using blood from 2 to 3 individuals with flow cytometry. Agonists and blood were added on the day of analysis. The reagent stability test was evaluated statistically using one-way ANOVA with Bonferronis post-hoc test. Results: The slight drop in percent positive platelets and median fluorescence intensity (MFI) seen over time in the reagent stability test was not statistically significant (p&gt;0,05). All platelet antibodies could be used after freezing/freeze-drying. The results are showing a declining trend, especially for MFI values. Conclusion: The frozen reagents used for platelet function testing is stabile up to 36 months. The results from the freeze-drying/freezing indicate that all platelet antibodies keep some activity but that some are more sensitive than others. Some antibodies could not be evaluated due to concentration of agonist in the sample being too low to induce activation. Due to individual variations further studies with more participants and more agonists in their optimal concentrations are needed. / Bakgrund: Hemostas är ett komplext system i kroppen som upprätthåller blodflödet och förhindrar blödning. Mukokutana blödningar kan uppstå hos patienter som har problem i den primära hemostasen vilket kan bero på trombocydefekter. På Klinisk kemi i Linköping mäts trombocytfunktion med flödescytometri. Trombocyterna aktiveras med olika agonister och svaret på stimuli mäts genom detektion av fluoroforkonjugerade antikroppar. Syftet med denna studie var att utvärdera långtidsstabiliteten av de frysta reagens som används för trombocytfunktionsutredningen, att testa om nya trombocytantikroppar klarar att frysas och att utvärdera reagensens hållbarhet för frystorkning. Metod: Antikroppar frystorkades/frystes i buffert och analyserades vid tre tillfällen med blod från 2 till 3 personer med flödescytometri. Agonist och blod tillsattes på analysdagen. Långtidsstabilitetstestet utvärderades statistiskt med  one-way ANOVA med Bonferronis post-hoc test. Resultat: En svag nedgång över tid i procent positiva trombocyter och MFI i långtidsstabilitetstestet var inte statistiskt signifikant (p&gt;0,05). Alla antikroppar gav signal efter frysning och frystorkning. Främst för MFI syns en nedåtgående trend över tid. Slutsats: Reagenset för trombocytfunktionsutredningen är stabilt upp till 36 månader. Resultat från frystorkningen tyder på att alla antikroppar klarar att frystorkas/frysas men vissa är känsligare än andra. Vissa antikroppar kunde inte utvärderas p.g.a. för låg agonistkoncentration för att inducera aktivering. Ytterligare försök måste göras med fler individer och fler agonister i optimal koncentration p.g.a. individuella skillnader i svar för agonister. Frystorkningsprocessen kan optimeras.

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While some rare and severe forms of platelet function disorders are now well characterized, many common types of platelet function disorders are not yet characterized. My hypothesis was that uncharacterized platelet function disorders that impair platelet function in aggregation and/or dense granule ATP release assays are associated with increased bleeding risk. The main goal of the thesis was to study the phenotype and bleeding risks for uncharacterized platelet function disorders, through analysis of the results from clinical laboratory tests of platelet function and for a detailed analysis of their reported bleeding symptoms. First, I assessed if lumi-aggregometry provides useful diagnostic information on platelet function and can be used to help decide if an individual has a bleeding disorder. Two cohorts of individuals were studied that had dense granule ATP release assessed in response to multiple agonists as part of a work-up for a bleeding disorder. Cohort I was comprised of individuals tested between January 2007 and June 2013 and cohort II was comprised of subjects tested at least twice by this assay prior to September 2015. Among subjects tested more than once for dense granule release defects as part of the work up for a bleeding disorder (cohort I; n=133; cohort II; n=17), normal findings with all tested agonists were often confirmed by the second test (cohort I: 83%; cohort II: 100%), but impaired release with multiple agonists was not often confirmed (cohort I: 34%; cohort II: 54%) and even if it was present, the finding was not predictive of a bleeding disorder. Consequentially, it was recommended that lumi-aggregometry should not be used to diagnose platelet function disorders. Next, I studied the bleeding risks associated with uncharacterized platelet function disorders, by evaluating subjects who had abnormal findings by validated assays, namely subjects who had defective aggregation responses to two or more agonists and/or dense granule deficiency. Bleeding history was evaluated using the International Society for Thrombosis and Haemostasis bleeding assessment tool (ISTH BAT) and the likelihood for bleeding symptoms/ problems, was estimated using odds ratios (OR) collected using the clinical history assessment tool - platelet (CHAT-P) for all affected subjects, a subgroup family with a mutation RUNX1, unaffected family v members and general population controls. Individuals with platelet function disorders (n=29) and the affected members of the family with the RUNX1 mutation (n=6) had elevated ISTH BAT scores (median: 9; range:0-18 and median: 8.5, range 4-15, respectively) and an increased risk of abnormal bruising (OR 15-65 and 11-67), nosebleeds (OR 23-40 and 19-121), menorrhagia (OR 6.5-29) and excessive bleeding after trauma or dental/surgical procedures (OR 9.5-44 and 15-77 ) and wound healing problems (OR 13 and 38) compared to general population control (n=60) and unaffected (n=12) family members. Overall, the platelet function disorders in the study present with a significantly increased risk of mild, rather than severe bleeding problems. These findings are important for individuals and healthcare providers to promote evidence-based care of common uncharacterized inherited platelet function disorders for individuals with RUNX1 mutations, dense granule deficiency and/or impaired aggregation responses. / Thesis / Master of Science (MSc) / Platelets are small blood cells that help stop bleeding. People who have platelets that do not work properly are more likely to bleed. Determining who has platelet problems can be challenging as there are limitations to diagnostic tests for these conditions. Additionally, the risks for bleeding in individuals with platelet problems are unknown. We looked at individuals with bleeding problems and found that a recommended test to assess platelet dense granule release, called lumi-aggregometry, wasn’t able to reliably identify persons with bleeding problems. Based on this, we recommend that lumi-aggregometry should not be used to diagnose platelet function disorders. We also found that individuals with uncharacterized platelet function disorders have increased risks for wound healing problems and experiencing bruising, nosebleeds, menorrhagia, and excessive bleeding after dental or surgical procedures. These risks are common among other mild bleeding disorders and will be important to differentiate bleeding risk from other platelet disorders.