<p> </p><p>Platelets play a key role in primary haemostasis but are also related to the pathogenesis of arterial thrombosis. Thrombin is the most effective agonist inducing platelet activation. Human platelets express two G-protein coupled thrombin receptors (GPCRs), called protease activated receptor (PAR)1 and PAR4. The aim of this study was to clarify differences in the activities of PAR1 and PAR4, especially focusing on their resistance towards the platelet inhibitor nitric oxide (NO) and their ability to undergo desensitization. For this, PAR1- and PAR4- activating peptides (APs) (SFLLRN and AYPGKF, respectively) were used. Different aspects of platelet activities were studied: aggregation and the rise in intracellular Ca²⁺ concentrations ([Ca²⁺]_i). Aggregation was analyzed with lumiaggregometry, and [Ca²⁺]_i were studied using the fura-2 method. PKC substrate phosphorylation and the expression of PAR1 surface receptors were also analyzed, using Western blot and flow cytometry, respectively. The results from this study showed that NO exerted similar inhibitory effects on the two thrombin receptors. However, PAR1 and PAR4 differed in their ability to undergo desensitization. In cumulative dose-response studies, a low concentration of PAR1-AP induced desensitization of platelets towards higher PAR1-AP concentrations. This was not the case when studying PAR4-AP. The mechanism behind the desensitization of PAR1 to some part involved PKC, at least when studying the mobilization of intracellular Ca²⁺. PAR1 desensitization did not seem to involve receptor internalization and neither did it affect the activity of PAR4. This thus suggests that PAR4 might be a more suitable therapeutic target in the future management of thrombosis.</p><p> </p>

CD109 is a 170kD glycosylphosphatidylinositol-anchored protein expressed on subsets of fetal and adult CD34+ haematopoietic stem cells, endothelial cells, activated T cells, and activated platelets. Cloning of the CD109 cDNA by our group identified the molecule as a novel member of the alpha2M/C3/C4/C5 family of thioester containing proteins. Curiously, CD109 bears features of both the alpha2M and complement branches of the gene family. Additionally CD109 carries the antigenic determinant of the Gov alloantigen system, which has been implicated in a subset of immune mediated platelet destruction syndromes. In this thesis, the status of CD109 in the evolution and phylogeny of the A2M family has been clarified. First, I elucidated the evolutionary relationships of CD109, and of the other eight human A2M/C3/C4/C5 proteins, using sequence analysis and a detailed comparison of the organization of the corresponding loci. Extension of this analysis to compare CD109 to related sequences extending back to placazoans, defined CD109 as a member of a distinct and archaic branch of the A2M phylogenetic tree. Second, in conjunction with collaborators, the molecular basis of the Gov alloantigen system was identified as an allele specific A2108C; Y703S polymorphism. Utilizing cDNA and genomic sequence we then developed methods to accurately and precisely genotype the Gov system. Finally, the expression kinetics of platelet CD109 was elucidated, in order to obtain basic information regarding its expression and subcellular localization, and to resolve discrepancies in reported platelet CD109 expression. Quantitative flow cytometry demonstrated that CD109 was expressed on the surface of activated platelets at very low levels in most healthy volunteers. In resting platelets, CD109 was localized to the OCS and intracellular storage granules. CD109 displayed differential agonist induced expression in comparison to GPIIb/IIIa epitope unmasking, and surface expression of CD62P and CD63. CD109 was rapidly expressed on the cell surface in response to low doses of both strong and weak agonists. This early expression is likely the result of CD109’s proximity to the plasma membrane in resting platelets. As such, CD109 is positioned to participate at early stages of primary haemostasis.

CD109 is a 170kD glycosylphosphatidylinositol-anchored protein expressed on subsets of fetal and adult CD34+ haematopoietic stem cells, endothelial cells, activated T cells, and activated platelets. Cloning of the CD109 cDNA by our group identified the molecule as a novel member of the alpha2M/C3/C4/C5 family of thioester containing proteins. Curiously, CD109 bears features of both the alpha2M and complement branches of the gene family. Additionally CD109 carries the antigenic determinant of the Gov alloantigen system, which has been implicated in a subset of immune mediated platelet destruction syndromes. In this thesis, the status of CD109 in the evolution and phylogeny of the A2M family has been clarified. First, I elucidated the evolutionary relationships of CD109, and of the other eight human A2M/C3/C4/C5 proteins, using sequence analysis and a detailed comparison of the organization of the corresponding loci. Extension of this analysis to compare CD109 to related sequences extending back to placazoans, defined CD109 as a member of a distinct and archaic branch of the A2M phylogenetic tree. Second, in conjunction with collaborators, the molecular basis of the Gov alloantigen system was identified as an allele specific A2108C; Y703S polymorphism. Utilizing cDNA and genomic sequence we then developed methods to accurately and precisely genotype the Gov system. Finally, the expression kinetics of platelet CD109 was elucidated, in order to obtain basic information regarding its expression and subcellular localization, and to resolve discrepancies in reported platelet CD109 expression. Quantitative flow cytometry demonstrated that CD109 was expressed on the surface of activated platelets at very low levels in most healthy volunteers. In resting platelets, CD109 was localized to the OCS and intracellular storage granules. CD109 displayed differential agonist induced expression in comparison to GPIIb/IIIa epitope unmasking, and surface expression of CD62P and CD63. CD109 was rapidly expressed on the cell surface in response to low doses of both strong and weak agonists. This early expression is likely the result of CD109’s proximity to the plasma membrane in resting platelets. As such, CD109 is positioned to participate at early stages of primary haemostasis.

Platelets play a key role in primary haemostasis but are also related to the pathogenesis of arterial thrombosis. Thrombin is the most effective agonist inducing platelet activation. Human platelets express two G-protein coupled thrombin receptors (GPCRs), called protease activated receptor (PAR)1 and PAR4. The aim of this study was to clarify differences in the activities of PAR1 and PAR4, especially focusing on their resistance towards the platelet inhibitor nitric oxide (NO) and their ability to undergo desensitization. For this, PAR1- and PAR4- activating peptides (APs) (SFLLRN and AYPGKF, respectively) were used. Different aspects of platelet activities were studied: aggregation and the rise in intracellular Ca2+ concentrations ([Ca2+]i). Aggregation was analyzed with lumiaggregometry, and [Ca2+]i were studied using the fura-2 method. PKC substrate phosphorylation and the expression of PAR1 surface receptors were also analyzed, using Western blot and flow cytometry, respectively. The results from this study showed that NO exerted similar inhibitory effects on the two thrombin receptors. However, PAR1 and PAR4 differed in their ability to undergo desensitization. In cumulative dose-response studies, a low concentration of PAR1-AP induced desensitization of platelets towards higher PAR1-AP concentrations. This was not the case when studying PAR4-AP. The mechanism behind the desensitization of PAR1 to some part involved PKC, at least when studying the mobilization of intracellular Ca2+. PAR1 desensitization did not seem to involve receptor internalization and neither did it affect the activity of PAR4. This thus suggests that PAR4 might be a more suitable therapeutic target in the future management of thrombosis.

Bileaflet mechanical heart valves (BMHVs) are prone to thrombus formation in the hinge region due to high shear stress combined with stagnation regions. This thesis research addresses the hypothesis that models that isolate and mimic BMHV hinge geometries can be used to quantitatively characterize procoagulant potential using a novel in vitro blood flow system. Furthermore, these results can be correlated with digital particle image velocimetry (DPIV) measurements detailing flow fields for the same models. The significant findings were that: 1) recalcification of recirculating citrated blood markedly increases the magnitude of thrombus forming reactions and the sensitivity for their detection; 2) platelet activation, and the presence of adequate platelet numbers are essential for the activation of coagulation under conditions of high shear; and 3) thrombin formation can be inhibited by blocking the platelet receptors that facilitate platelet aggregation. The DPIV studies give some insight into why different channel geometries resulted in varying propensities for coagulation. The channel geometries with abrupt changes in diameter induced significantly higher levels of TAT and also formed jets that were subject to increased entrainment of the stagnant fluid in the chamber. This entrainment enables more mixing of the shear-activated platelets with the surrounding flow, which can propagate the coagulation cascade, thus increasing the chance for thrombus formation. The influence of abrupt changes in diameter was also evident in the BMHV human blood studies. The MP valve, which has a tortuous hinge pathway, induced significantly more TAT formation than the SJM Standard valve with a smoother hinge channel. Thus, BMHV hinge geometry should be as smooth and free of diameter changes as possible to eliminate stagnation regions that enable activated platelets to congregate and propagate the coagulation cascade. Leakage gap width also had a significant effect not only on procoagulant potential but also on platelet activation. Both the low and high leaker prototype valves had significantly higher levels of platelet activation compared to the SJM Standard valve, but only the low leaker valve demonstrated a higher propensity for coagulation. Thus, to minimize both platelet activation and thromboemboli formation, an optimal gap width should be maintained for BMHVs.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2008. / Härtill 5 uppsatser. Includes bibliographical references.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Platelets play an important role in the haemostatic process in order to seal damaged blood vessels. The platelets form a platelet plug at the damaged area and prevent blood loss. Once the damage to the vessel wall has been covered, the platelets retract the coagulum, to allow the blood to flow freely in the vessel. Free oscillation rheometry (FOR) can be used for analysis of coagulation as measured by clotting time and changes in clot elasticity (G'). Clot G' provides information about the fibrin network in the coagulum and the platelets’ ability to retract the coagulum. FOR analysis is performed using the ReoRox® 4 instrument. The blood sample is added to a cylindrical sample cup, which is set into free oscillation. The frequency and damping of the oscillation is recorded over time as the blood coagulates. The change in G' is calculated from the frequency and damping measured. Patients with malignant haematological diseases are often thrombocytopaenic and require platelet transfusions to prevent or stop bleeding. To ensure good haemostatic function in the recipient it is important that the quality of the platelets used for transfusion is well preserved. The aim of this thesis was to determine the quality of platelet concentrates (PCs), during storage, using various in vitro methods, including FOR, and to investigate how various preparation processes affect the quality. We also investigated whether FOR can be used to evaluate the haemostatic status in subjects at risk for thrombosis or bleeding as well as how the haemostatic status was affected by a platelet transfusion. We show that FOR can provide information about the coagulation properties in subjects at risk of thrombosis (pregnant women) or bleeding (thrombocytopaenic patients). We also show that the coagulation as measured by FOR is influenced by red blood cells and the fibrinogen concentration. However, the presence of functional platelets accounted for 90% of the G'. Furthermore we present data that FOR can provide information on the haemostatic effect of platelet transfusions and on the function of the transfused platelets. PCs produced by two different cell separators showed similar quality during storage for 7 days as assessed by FOR analysis. Leukocytes in the PCs can cause transfusion-associated graft-versus-host disease which can be prevented by gamma irradiation of the PCs. Gamma irradiation did not affect the quality of PCs during 7 days of storage as analysed by FOR. The clotting time was unchanged during the storage period. The capacity of platelets to retract the coagulum was reduced from days 1 to 5 of storage as seen by a prolonged time to reach maximum G' and the reduced mean change in G' per minute. However, if sufficient time is allowed for the platelets to regain their function, the clot will be fully retracted (as seen by a well maintained maximum G'). The FOR parameters were similar for 5- and 7-day old PCs, which, combined with other in vitro tests (e.g. hypotonic shock response, changes in pH, swirling, lactate and glucose), support the prolongation of the platelet storage period to 7 days. Intercept™ treatment of PCs can be performed to inhibit replication of contaminating bacteria in PCs. Intercept™ treatment of PCs did not diminish the clot-promoting capacity of the platelets as assessed by FOR clotting time. In conclusion, FOR is a promising method for assessing hyper- and hypocoagulability. It can provide information on the haemostatic effect of platelet transfusions and the function of the transfused platelets. FOR was also shown to be useful for analysing PC quality during different preparation and storage conditions.

No description available.

Platelets are small fragments, but they are of crucial importance for the coagulation. The risk of spontaneous bleeding increases when the level of platelets falls below a thrombocyte particle concentration threshold value of 50 x 109/L. In those cases a platelet transfusion might be compulsory. Ongoing research tries to improve the quality of the platelets and to increase the safety of the method used. However, we still need to better understand which factors that affect how patients react upon platelet transfusion. In this study, 100 transfusions performed at Uppsala University Hospital during 2009 were examined. The platelets used had been produced with apheresis followed by pathogen inactivation by Intercept Blood SystemTM. Platelets were counted before and after transfusions and the increase was calculated in purpose to examine how well the patients responded to the platelet transfusions. These values were plotted against platelet storage time in order to examine the possible impact on the result of treatment.