Title of Diploma Thesis: Production and use of transfusion products in the District Hospital Trutnov. Aim of the work: The aim was to evaluate the production of transfusion products (TP) in the period 2014-2019 at the Transfusion and Hematology Department (THO) in the District Hospital Trutnov and their use for hemotherapy by individual medical disciplines. Futhermore, to compare the production of TP within the Czech Republic together with selected European countries. The incidence of post-transfusion reactions and other adverse events during the reporting period is also included. Material and methods: The methodological part describes the standard procedures of pre- transfusion examination using the column agglutination method from the company Grifols, S.A., laboratory examination of donors and transfusion products manufactured at the Transfusion Department Trutnov. Conclusion: Despite all the recommendations and efforts to reduce the use of transfusion products, this condition has not been proven both in our department, within the Czech Republic and in Europe. This work recommends deleucotization of transfusion products for the national and European part, unification of procedures for transfusion in a given disease, mandatory introduction of NAT testing to minimize the risk of transmission...

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Haemostasis is maintained by a dynamic balance between pro- and anti-thrombotic mediators. Its dysregulation can lead to bleeding or thrombosis, and is a major cause of morbidity and mortality. Thus, elucidation of the mechanisms involved in maintaining or disrupting this balance have important implications in health and disease. Investigative tools enable characterization of the haemostatic system, but are often associated with limitations. For instance, haemostasis in animal models is often investigated by assessing bleeding responses in one particular vessel or tissue without a complete understanding of how the results translate to the regulation of haemostasis in other vascular beds. As a second example, microparticles (MPs) are a heterogeneous population of submicron-sized vesicles that may be important in thrombosis. With the exception of a few subtypes, MPs cannot be reliably characterized using widely accessible techniques. Finally, the thrombin generation assay (TGA), which measures ex vivo activation and inhibition of thrombin, is a promising tool for clinical assessment of thrombosis and haemostasis. However, characterization of thrombin generation in the general population, and the development of point of care testing are in their infancies. As a result, the TGA remains largely a research tool. The works described in this thesis specifically seek to address these three limitations in thrombosis and haemostasis research. The first isolated murine arterial bleeding model is presented and its characterization with respect to bleeding in other vascular tissues is described. In addition, a solid-phase capture assay for evaluating procoagulant, P-selectin-binding MPs, which are postulated to be mediators of thrombosis, was developed in order to determine whether these MPs associate with risk of recurrent venous thromboembolism. Lastly, a 25 x 20 mm chip that performs four individual thrombin generation assays using ~10 µl of capillary blood was developed as a proof of concept for point of care thrombin generation testing. / Thesis / Doctor of Philosophy (PhD)

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.

Recent studies suggest that factor (F) XII plays a key role in thrombus stabilization and growth but is dispensable for hemostasis. We have previously shown that histidine-rich glycoprotein (HRG), a protein present in platelets and plasma, binds FXIIa and inhibits FXII autoactivation and FXIIa-mediated activation of FXI, thereby downregulating thrombosis. HRG binds various ligands, including FXIIa, fibrin(ogen), nucleic acids and polyphosphate (polyP). Studies have shown that polyP, released from activated platelets, and artificial surfaces like catheters, can promote FXII activation. This suggests that HRG can downregulate the activation of the contact system. This thesis aims to determine the potential mechanisms by which HRG modulates platelet function and thrombosis induced by polyP or catheters. We show that HRG binds polyP with high affinity and inhibits the procoagulant, prothrombotic and cardiotoxic effects of polyP via at least two mechanisms. First, HRG binds polyP and neutralizes its procoagulant activities and cytotoxic effects. Second, HRG binds FXIIa and attenuates its capacity to promote autoactivation and activate FXI. Also, we identify that HRG serves as a molecular brake for the contact system by attenuating the procoagulant activity of FXIIa regardless of whether FXII activation is triggered systemically with polyP or occurs locally on the surface of catheters. Our studies have identified HRG as a novel ligand for platelet receptor GPIbα on resting platelets, and upon activation, it competes with fibrinogen for binding to GPIIb/IIIa integrin, thereby inhibiting platelet aggregation. These findings suggest that HRG may modulate coagulation as well as platelet function. Therefore, supplementation with HRG or HRG analogs may serve as a potential therapeutic option to attenuate polyP or catheter-induced thrombosis without perturbing hemostasis. / Dissertation / Doctor of Philosophy (PhD)

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<p>The management of thrombosis in the presence of thrombocytopenia is challenging because the inherent risk of bleeding associated with anticoagulant use may increase due to low platelet counts. Guidelines regarding anticoagulant use in this situation are based mainly on expert opinions and anecdotal data. We developed an <em>in-vitro</em> model to study the effect of anticoagulants on plasma clot formation in the presence of low platelet counts. We used thromboelastography (TEG) to measure global viscoelastic properties of clot formation and scanning electron microscopy (SEM) to observe and quantify changes in the fibrin clot structure. Experiments were conducted in plasma with varying platelet concentrations from <10 >– 150 × 10⁹/L. Clotting was activated with tissue factor (TF) and calcium, in the presence of factor XIIa inhibitor, corn trypsin inhibitor. One of the following anticoagulants at therapeutic concentration was added to the mixture: unfractionated heparin (UFH), dalteparin, fondaparinux, rivaroxaban or dabigatran. We found clotting had different sensitivity to TF concentration depending on the anticoagulant present. Effects on TEG parameters varied at a fixed TF concentration with each anticoagulant. UFH had the greatest influence, delaying clotting significantly at low platelet counts. The factor-specific anticoagulants had the least impact on TEG parameters. SEM revealed that UFH had the greatest impact on clot structure. UFH caused significant increase in porosity and fibrin widths and had significantly less fibers when platelets decreased. In conclusion, this study may provide fundamental data to understand clot formation in the presence of anticoagulants at low platelet counts. At low platelets the anticoagulants can jeopardize clot formation, especially UFH. The mechanism of each anticoagulant may contribute to the variation in response to TF initiated clotting. AT-dependent anticoagulants compromised plasma clotting more than the newer factor specific anticoagulants, possibly related to the multiple, non-specific inhibition of coagulation factors.</p> / Master of Science (MSc)

Cardiovascular diseases are a major cause of mortality and morbidity in the developed countries. Anti-platelet therapy is a cornerstone treatment for patients with cardiovascular diseases. Patients are routinely managed with a combination therapy consisting of aspirin and clopidogrel. Aspirin inhibits cyclooxygenase 1 (COX 1) a crucial intermediate enzyme involved in thromboxane biosynthesis. Clopidogrel on the other hand antagonizes ADP receptor P2Y12. ADP is a weak platelet agonist stored in platelet dense granules and is released upon platelet activation. ADP activates platelets through two purinergic receptors namely P2Y1 and P2Y12 these receptors couple to Gq and Gi class of G-proteins, respectively. P2Y1 causes calcium mobilization through activation of PLC-β. P2Y12 inhibits adenylyl cyclase, causes activation of Rap1B and Akt. Signaling from both the receptors is required for complete integrin activation, thromboxane generation and Erk activation. Previous studies have shown that P2Y12 potentiates fibrinogen receptor activation, secretion, thrombi stabilization, thrombin generation, platelet leukocyte aggregation formation. ThromboxaneA2 (TXA2) is a potent platelet agonist generated through arachidonic acid metabolism in platelets. TXA2 thus, generated after platelet activation acts as a positive feedback mediator along with ADP. Under physiological conditions, platelet activation leads to thrombin generation through coagulation cascades. Generated thrombin activates PAR receptors and ADP is released from dense granules, which further potentiates thromboxane generation downstream of PARs. Current anti-platelet therapy regimens often include P2Y12 antagonists and aspirin in management of patients with acute coronary syndrome (ACS) and in those undergoing percutaneous coronary intervention (PCI) with stent implantation. However, there still exists a need for improved treatment strategies as not all patients benefit from this dual combination therapy. Reasons include, poor responders either to P2Y12 antagonists or to aspirin, or if aspirin is contraindicated in these patient populations. In the current study we evaluated the role of P2Y12 in thromboxane generation under physiological conditions. We studied serum thromboxane generation in a model system wherein P2Y12 was antagonized or deficient. Using pharmacological approaches we show that dosing mice with 30mg/Kg/body weight clopidogrel or 3mg/Kg/body weight prasugrel decreased serum thromboxane levels when compared to the control mice. Pre-treatment of human blood ex vivo with active metabolites of clopidogrel (R361015) or prasugrel (R138727) also led to reduction in thromboxane levels. We also evaluated serum thromboxane levels in P2Y receptor null mice, serum thromboxane levels in P2Y1 null mice were similar to those in wild type littermates, and were inhibited in P2Y12 null mice. Furthermore, serum thromboxane levels in P2Y12 deficient patients, previously described in France and Japan, were also evaluated and these patients had lower serum thromboxane levels compared to normal controls. In a pilot study, serum thromboxane levels were radically reduced in healthy human volunteers upon dosing with clopidogrel, compared to the levels before dosing. In conclusion, P2Y12 antagonism alone can decrease physiological thromboxane levels. Thus P2Y12 regulates physiological thromboxane levels. Further it is known that ADP-induced thromboxane generation is integrin-dependent. However it is not clear if other potent platelet agonists like thrombin require outside-in signaling for thromboxane generation. Our results show that thrombin-induced thromboxane generation was independent of integrins i.e. when platelets were stimulated with PAR agonists in presence of fibrinogen receptor antagonist thromboxane generation was not affected. Since PAR agonists, unlike ADP, activate G12/13 signaling pathways. Hence, we hypothesized that these pathways might play a role in TXA2 generation. Our results show, that inhibition of ADP-induced thromboxane generation by fibrinogen receptor antagonist SC57101 was rescued by costimulation of G12/13 pathways with YFLLRNP. This observation suggested an existence of a common signaling effector downstream of integrins and G12/13 pathways. Next, we evaluated role of three potential tyrosine kinases; c-Src, Syk and FAK (Focal Adhesion Kinase) that are known to be activated by integrins. Our results showed that c-Src and Syk kinase did not play a role in ADP-induced functional responses in platelets. We observed differential activation of FAK downstream of integrins and G12/13 pathways. ADP-induced activation of FAK was integrindependent and SFK-independent. On the other hand selective activation of G12/13 pathway lead to FAK activation, in SFK and Rho dependent manner. We also evaluated specificity of new FAK inhibitor TAE-226 to understand the role of FAK in TXA2 generation. Our results showed that TAE-226 exhibited non-specific effects at higher concentrations. Furthermore, in comparison to WT mice, FAK null mice did not show any difference in TXA2 generation. Therefore, we concluded that differential activation of FAK occurs downstream of Integrins and G12/13 pathways. However, the common effector molecule downstream of integrins and G12/ 13 pathways contributing to TXA2 generation in platelets remains elusive. / Molecular and Cellular Physiology

Protein Kinase C delta (PKCδ) is expressed in platelets and activated downstream of protease-activated receptors (PAR)s and glycoprotein VI (GPVI) receptors. We evaluated the role of PKCδ in platelets using two approaches - pharmacological and molecular genetic approach. In human platelets pretreated with isoform selective antagonistic RACK peptide (δV1-1)TAT, and in the murine platelets lacking PKCδ, PAR4-mediated dense granule secretion was inhibited, whereas GPVI-mediated dense granule secretion was potentiated. These effects were statistically significant in the absence and presence of thromboxane A2 (TXA2). Furthermore, TXA2 generation was differentially regulated by PKCδ. However, PKCδ had a small effect on platelet P-selectin expression. Calcium- and PKC-dependent pathways independently activate fibrinogen receptor in platelets. When calcium pathways are blocked by dimethyl-BAPTA, AYPGKF-induced aggregation in PKCδ null mouse platelets and in human platelets pretreated with (δV1-1)TAT, was inhibited. In a FeCl3-induced injury in vivo thrombosis model, PKCδ-/- mice occluded similar to their wild-type littermates. Hence, we conclude that PKCδ differentially regulates platelet functional responses such as dense granule secretion and TXA2 generation downstream of PARs and GPVI receptors, but PKCδ deficiency does not affect the thrombus formation in vivo. We further investigated the mechanism of such differential regulation of dense granule release by PKCδ in platelets. SH2 domain-containing Inositol Phosphatase (SHIP)-1 is phosphorylated on Y1020, a marker for its activation, upon stimulation of human platelets with PAR agonists, SFLLRN and AYPGKF, or GPVI agonist, convulxin. GPVImediated SHIP-1 phosphorylation occurred rapidly at 15 sec whereas PAR-mediated phosphorylation was delayed, occurring at 1 min. Lyn and SHIP-1, but not SHIP-2 or Shc, preferentially associated with PKCδ upon stimulation of platelets with a GPVI agonists, but not with a PAR agonist. In PKCδ null murine platelets, convulxin-induced SHIP-1 phosphorylation was inhibited, suggesting that PKCδ regulates the phosphorylation of SHIP-1. Furthermore, in Lyn null murine platelets, GPVI-mediated phosphorylations on Y-1020 of SHIP-1, Y311 and Y155 of PKCδ were inhibited. In murine platelets lacking Lyn, or SHIP-1, GPVI-mediated dense granule secretions were potentiated, whereas PAR-mediated dense granule secretions were inhibited. Phosphorylated SHIP-1 associated with phosphorylated-Y155 PKCδ peptide. Therefore, we conclude that Lyn-mediated phosphorylations of PKCδ and SHIP-1 and their associations negatively regulate GPVI-mediated dense granule secretion in platelets. / Physiology