Neonatal alloimmune thrombocytopenia (NAIT) is caused by maternal alloantibodies against fetal platelet antigens inherited from the father and which are absent from maternal platelets. In Caucasians, antibodies against the Leu33 (HPA-1a) polymorphism of integrin β3 (part of the platelet αIIbβ3 complex) account for >70% of cases. Antenatal screening for these antibodies does not currently take place in the UK, partly because of the absence of sensitive, predictive tests. We hypothesized that the poor sensitivity and predictive abilities of current assays are due to the use of β3 in an inappropriate conformation, resulting in sub-optimal binding of HPA-1a antibodies. We hypothesized firstly that in vitro induced changes to αIIbβ3 might alter accessibility of the HPA-1a epitopes to alloantibodies, thus reducing assay sensitivity. Secondly, we hypothesized that HPA-1a antibodies are stimulated by, and preferentially recognise, β3 in association with αv, a molecule present on placental syncytiotrophoblasts, and that reactivity against platelet αIIbβ3 reflects only cross-reactivity with αvβ3. Our first hypothesis was proven by demonstrating that use of the cation chelating compound EDTA, used by many diagnostic laboratories as a component of assay reagents or present in blood samples as anticoagulant, resulted in significantly reduced assay sensitivity. These findings were confirmed in an international workshop. Support for our second hypothesis was provided by demonstrating enhanced reactivity of a small panel of examples of anti-HPA-1a against αvβ3 compared to αIIbβ3 and by molecular modelling data. We also showed that HPA-1a antibodies can inhibit platelet function by using a novel application of the ROTEM® delta thromboelastograph and an immunofluorescence assay in which we demonstrated blocking of platelet function using a monoclonal antibody, PAC-1, that binds only to activated αIIbβ3. These studies provide possible explanations for the poor sensitivity and predictive abilities of current assays and suggest further areas for research.

Haematological malignancies and their treatment lead to prolonged periods of severe thrombocytopenia (platelet count ≤ 50 x 10⁹/l). Despite the use of prophylactic platelet transfusions, haemorrhage remains an important complication during this thrombocytopenic period. Within a 30 day period up to 70% of patients have clinically significant haemorrhage (World Health Organization (WHO) grade 2 or above bleeding) and up to 10% have severe or life-threatening haemorrhage (WHO grade 3 or 4 bleeding). Hence our current management of these patients to prevent haemorrhage is sub-optimal. The aim of this thesis was to identify clinical and laboratory factors that may predict the risk of haemorrhage in patients with haematological malignancies and severe thrombocytopenia. This was achieved via several different study designs and assessed the effect of clinical and laboratory factors on any or clinically significant haemorrhage and their effect on intracranial haemorrhage. This thesis has demonstrated that there is no consensus on how bleeding is assessed and graded in this patient group. Also it showed that the absolute immature platelet number may be a better alternative to the total platelet count to guide administration of platelet transfusions. Female sex, a previous history of a fungal infection, a high C-reactive protein, a high white cell count, a low platelet count, anaemia, impaired renal function, and recent clinically significant haemorrhage were all found to be independent risk factors for haemorrhage. Patients who were in complete remission from their haematological malignancy had a much lower risk of bleeding.

Lysophosphatidic acid (LPA) belongs to a class of extracellular lipid signaling molecules. In the vasculature, LPA may regulate platelet activation and modulate endothelial and smooth muscle cell function. LPA has therefore been proposed as a mediator of cardiovascular disease. The bulk of circulating LPA is produced from plasma lysophosphatidylcholine (LPC) by autotaxin (ATX), a secreted lysophospholipase D (lysoPLD). Early studies suggest that some of the production of circulating LPA is platelet-dependent. ATX possesses an N-terminal somatomedin B-like domain suggesting the hypothesis that ATX interacts with platelet integrins which may localize ATX to substrate in the membrane and/or alter the catalytic activity of ATX. Using static adhesion and soluble binding assays we found that ATX does indeed bind to platelets and cultured mammalian cells in an integrin-dependent manner which is blocked by integrin function-blocking peptides and antibodies. This binding increases both the activity of ATX and localization of its product, LPA, to the platelet/cell membrane. LPA is generally stimulatory to human platelets although platelets from a small population of donors are refractory to LPA stimulation. Likewise LPA is inhibitory to murine platelets. We previously found that LPA receptor pan-antagonists reduce agonist-induced platelet activation, and partial stimulation of LPA5 specifically increases platelet activation in humans. Since both LPA5 and LPA4 are present at significant levels in human platelets, we hypothesized that LPA4 is responsible for an inhibitory pathway and LPA5 is responsible for an inhibitory pathway. We used mice deficient in LPA4 to test this model. Isolated platelet function tests revealed no major difference between lpa4-/- mice compared with WT mice although lpa4-/- mice were more prone to FeCl3-induced thrombosis. Paradoxically, chimeric mice reconstituted with lpa4-/- deficient bone marrow derived cells were protected from thrombosis. These discrepancies may be explained by involvement of endothelial cells and the relative scarcity of LPA receptors in murine platelets compared with human platelets. Taken together, these results demonstrate two critical regulators of LPA signaling and open up new avenues to further our understanding of atherothrombosis.

Platelet secretion is important for hemostasis and thrombosis. The components released are also involved in atherosclerosis, inflammation, angiogenesis, and tumor growth. Though the exact mechanism(s) of platelet secretion is still elusive, accumulating evidence demonstrates that SNAREs (Soluble N-ethylmaleimide Sensitive Factor Associated Receptor) and their regulatory partners are critical for platelet exocytosis. Formation of a trans-bilayer complex composed of one v-SNARE (i.e. VAMPs) and two t-SNAREs (i.e. syntaxin and SNAP-25-type) is minimally required for membrane fusion. Regulatory proteins control the rate and specificity of the complex assembly. VAMP-8 and SNAP-23 (a SNAP-25-type t-SNARE) are clearly important; however, the identity of the functional syntaxin has been controversial. Previous studies, using anti-syntaxin antibodies in permeabilized platelets, suggested roles for both syntaxin-2 and -4. These conclusions were experimentally tested using platelets from syntaxin knockout mice and from a Familial Hemophagocytic Lymphohistiocytosis type 4 (FHL4) patient that lacks syntaxin-11. Platelets from syntaxin-2 and syntaxin-4 single or double knockout mice had no significant secretion defect. However, platelets from the FHL4 patient had a robust defect, though their morphology, activation, and cargo levels appeared normal. Semi-quantitative western blotting showed that syntaxin-11 is the most abundant syntaxin in both human and murine platelets. Co-immunoprecipitation experiments showed that syntaxin-11 forms SNARE complexes with VAMP-8 and SNAP-23. These data conclusively demonstrate that syntaxin-11, but not syntaxin-2, or -4, is required for platelet exocytosis. We also show that a syntaxin binding protein, tomosyn-1, is important for platelet exocytosis and hemostasis. Tomosyn-1 was identified from platelet extracts using affinity chromatography, RT-PCR analysis, and western blotting analysis. Tomosyn-1 was co-immunoprecipitated with syntaxin-11/SNAP-23 from both resting and activated platelet extracts. Platelets from tomosyn-1-/- mice displayed a secretion defect, but their morphology and activation appeared normal. Tomosyn-1-/- mice showed impaired thrombus formation in two different injury models. Given the importance of platelet secretion to hemostasis, it is hoped that the insights gained from these studies in this dissertation will help to identify new and more valuable therapeutic targets to control clot formation.

In addition to their role in hemostasis, platelets appear to contribute to vascular inflammatory diseases. Platelets achieve this through the secretion of various prothrombotic and pro-inflammatory molecules. Platelet secretion is mediated by integral membrane proteins called Soluble NSF Attachment protein REceptors (SNAREs). SNAREs come from both granule/vesicle membranes (v-SNAREs) and target membranes (t-SNAREs) to form a trans-bilayer complex that promotes membrane fusion and subsequent granule cargo release. The work described in this dissertation dissects various, yet related aspects of platelet secretion in both physiological relevant and pathological circumstances. Atherosclerosis is a leading cause of death in the westernized countries and a major contributor to heart attacks and strokes. Given the potential involvement of platelets in atherosclerosis and previous work from our laboratory showing that VAMP-8 is the primary v-SNARE for platelet secretion, one part of this dissertation focuses on the role of VAMP-8- mediated secretion in atherosclerosis. The data showed that the deletion of VAMP-8 in the ApoE-/- null model of chronic atherosclerosis attenuated plaque development compared to the wild type littermates. Aged (50 week) VAMP-8-/-/ApoE-/- mice showed a reduction in lesion size compared to ApoE-/- controls, as measured by Oil Red-O staining of the plaques in the aortic sinus and by en face analysis of plaques in the aortic arch. These data show that the loss of VAMP-8 attenuates the development of atherosclerotic plaques and suggest that platelet secretion contributes to atherosclerosis. Considering the vital role of platelet secretion in both physiological and pathological conditions, it is essential to understand how it is regulated. SNARE proteins are controlled by a range of regulatory molecules that affect where, when, and with whom they form trans-bilayer complexes for membrane fusion. One family of such regulators is the Munc18 family: platelets contain three (Munc18a-c). The second part of this dissertation focuses on the role of Munc18b/STXBP2. Mutations in the Munc18b/STXBP2 gene underlie Familial Hemophagocytic Lymphohistocytosis type 5 (FHL5), which is a life- threatening disease caused by dysregulation of the immune system. Platelets from two biallelic FHL5 patients had almost undetectable levels of Munc18b/STXBP2 levels; the levels of Munc18a increased slightly and Munc18c levels were unaffected. Syntaxin 11 levels were affected but the levels of other secretory machinery proteins were normal. Platelet secretion from dense and alpha granule in two biallelic patients and the one heterozygous patient was decreased. The release of serotonin from dense granules, and platelet factor 4 (PF4) from alpha granules was profoundly affected in the biallelic patients and partially affected in the heterozygote heterozygous patient. Lysosome release was affected only from the platelets of the biallelic patients. These data indicate that Munc18b plays a key role in platelet secretion. Ras is the prototypical member of a family of low molecular weight, GTP-binding proteins. It affects various cellular functions by cycling between an active, guanine triphosphate (GTP) and an inactive guanine diphosphate (GDP) -bound state. Little is known about the role of Ras activation in platelets. The third part of this dissertation focuses on what could be learned about Ras’ role by analyzing platelets from patients with Noonan Syndrome. Specific mutations in the genes encoding elements of Ras signaling pathways are associated with Noonan Syndrome. Platelets from Noonan Syndrome patients with a mutation in kRas (F156V) were analyzed and shown to have a defect in aggregation in response to low levels of agonist. These data suggest that Ras may play a functionally relevant role in platelet activation. In summary, the experiments presented in investigations of this dissertation support a role for platelet secretion in several pathological conditions and suggest that platelet secretion assays may serve as useful as diagnostic tools for some genetic diseases. In addition, these studies elucidate the importance of understanding the regulation of platelet exocytosis, to drive the development of new antithrombotic therapeutics.

Hypercholesterolemia is a dominant risk factor for a variety of cardiovascular diseases and involves a chronic inflammatory component in which neutrophil activity plays a critical role. Recently, fluid shear stress mechanotransduction has been established as a control mechanism that regulates the activity of neutrophils by reducing the formation of pseudopods and the surface expression of CD18 integrins, thereby rendering these cells rounded, deformable, and non-adhesive. This is critical for maintaining a healthy circulation, because chronically activated neutrophils not only release excess cytotoxic and degradative agents but also exhibit a reduced efficiency to pass through the small vessels of the microcirculation leading to increased microvascular resistance. We hypothesized that aberrant neutrophil mechanosensitivity to fluid shear stress due to the altered blood environment (i.e., excess plasma cholesterol) is a contributing factor for elevated hemodynamic resistance in the microcirculation associated with hypercholesterolemia. For this purpose, the present work firstly showed that the sensitivity of neutrophils to fluid shear stress depends on the cholesterol-dependent fluidity of the cell membrane, and that, in the face of hypercholesterolemia, the neutrophil mechanosensitivity highly correlated with the plasma levels of free cholesterol. The second part of this project demonstrated that, when subjected to shear stress fields, leukocyte suspensions exhibited transient (within 10 min of flow onset) time-dependent reductions in their apparent viscosity. Moreover, shear-induced changes in viscosity of cell suspensions were influenced by disturbances of membrane cholesterol and fluidity in a fashion similar to that for shear-induced pseudopod retraction. Finally, the third part of this work provided evidence that neutrophils played a role in hypercholesterolemia-related impairment of flow recovery response to transient ischemia. In conclusion, results of the current work provided the first evidence that cholesterol is an important component of the neutrophil mechanotransducing capacity and impaired neutrophil shear mechanotransduction may disturb the blood flow rheology, leading to elevations in the apparent viscosity as well as in the resistance. This cholesterol-linked perturbation may be a contributing factor for the pathologic microcirculation associated with hypercholesterolemia.

Platelet exocytosis is essential for hemostasis and for many of its sequelae. Platelets release numerous bioactive molecules stored in their granules enabling them to exert a wide range of effects on the vascular microenvironment. Are these granule cargo released thematically in a context-specific pattern or via a stochastic, kinetically-controlled process? My work describes platelet exocytosis using a systematic examination of platelet secretion kinetics. Platelets were stimulated for increasing times with different agonists (i.e. thrombin, PAR1-agonist, PAR4-agonist, and convulxin) and micro-ELISA arrays were used to quantify the release of 28 distinct α-granule cargo molecules. Agonist potency directly correlated with the speed and extent of release. PAR4-agonist induced slower release of fewer molecules while thrombin rapidly induced the greatest release. Cargo with opposing actions (e.g. pro- and anti-angiogenic) had similar release profiles, suggesting limited thematic response to specific agonists. From the release time-course data, rate constants were calculated and used to probe for underlying patterns. Probability density function and operator variance analyses were consistent with three classes of release events, differing in their rates. The distribution of cargo into these three classes was heterogeneous suggesting that platelet secretion is a stochastic process potentially controlled by several factors such as cargo solubility, granule shape, and/or granule-plasma membrane fusion routes. Sphingosine 1 phosphate (S1P) is a bioactive lipid that is stored in platelets. S1P is essential for embryonic development, vascular integrity, and inflammation. Platelets are an abundant source of S1P due to the absence of the enzymes that degrade it. Platelets release S1P upon stimulation. My work attempts to determine how this bioactive lipid is released from platelets. Washed platelets were stimulated with agonists for defined periods of time and the supernatant and pellet fractions were separated by centrifugation. Lipids were separated by liquid phase extraction and S1P was quantified with a triple quadrapole mass spectrometer. A carrier molecule (BSA) is required to detect release of S1P. Further, there is a dose-dependent increase in total S1P with increasing BSA. S1P release shows characteristics similar to other platelet granule cargo e.g. platelet factor IV (PF4). Platelets from Unc13-d Jinx mice and VAMP8-/- mice, which are secretion-deficient (dense granule, alpha granule and lysosome), were utilized to understand the process of S1P release. S1P release was more affected in Unc13-d Jinx mice mirroring their dense granule secretion defect. Fluorescence microscopy and sub-cellular fractionation were used to examine localization of S1P in platelets. S1P was observed to be enriched in a granule population. These studies indicate the existence of two pools of S1P, a readily extractable agranular pool, sensitive to BSA, and a granular pool that requires the secretion machinery for release. The secretion machinery of platelets in addition to being involved in the release of normal granule cargo is thus proved to be involved in the release of bioactive lipid molecules like S1P.

<p>Myocardial infarction (MI) is often caused by a disruption of an atherosclerotic plaque with activation of coagulation, platelets and inflammation. The aims were; to investigate whether the oral direct thrombin inhibitor, ximelagatran affected markers for coagulation, platelet and inflammation in a patient cohort with recent MI and if the coagulation markers could identify patients with increased risk of new ischemic events; to evaluate some of the mechanisms involved in formation of platelet-monocyte aggregates (PMAs). </p><p>In a biomarker substudy patients with recent MI were randomized to 24-60 mg of ximelagatran or placebo for six months. There was a persistent dose-independent reduction of coagulation markers (F1+2, D-dimer) by ximelagatran treatment. 60 % reduced their D-dimer levels after one week and that group had less ischemic events during treatment. There was an early increase of the platelet activation marker and ximelagatran in higher doses attenuated these increased levels. Both in vivo and in vitro the direct thrombin inhibitor diminished procoagulant activity and tissue factor (TF) presenting microparticles. In contrast, the inflammatory markers increased after six months of ximelagatran treatment. The PMA-levels were elevated for long-term after MI. In vitro thrombin inhibition diminished formation of PMAs. Formation of PMAs in stimulated whole blood was P-selectin dependent and induced TF expression through phosphorylation of the Src-family member Lyn in monocytes.</p><p>Addition of an oral direct thrombin inhibitor reduces coagulation and platelet activation markers for long-term after a MI together with reduced procoagulant activity which may contribute to the clinical benefit of the drug. Early reduction of D-dimer levels seems to be suitable to identify patients with reduced risk of new ischemic events independent of antithrombotic treatment. Circulating PMAs persist after a MI connecting coagulation to inflammation. Within these aggregates P-selectin induces TF, the main initiator of coagulation, partly through phosphorylation of Lyn.</p>

Inherited disorders of platelet function are a rare and heterogeneous group of diseases usually characterised by a mild to moderate bleeding tendency. Typical bleeding symptoms are easy bruising, epistaxis, menorrhagia as well as mucocutaneous and perioperative bleeding. The performance of platelet function diagnostics in children is hampered by age-dependent restriction of blood sample size, poor venous access, and the lack of reproducible test reference ranges for children of different age groups. Platelet function testing is limited to specialised centres, because platelet function test procedures are complicated and time-consuming, which most likely results in a relevant number of undiagnosed and incorrectly classified children with clinically relevant platelet function defects. Evaluation of bleeding history and bleeding symptoms is essential for a rational step-bystep approach to diagnosis. Platelet function diagnostics should be preceded by the exclusion of thrombocytopenia, von Willebrand disease, and secondary haemostasis defects. Light transmission aggregometry is still considered the standard for the assessment of platelet function. Every effort should be made to classify the specific platelet function defect in the patient, because this is essential for accurate treatment and counselling. / Angeborene Thrombozytenfunktionsstörungen stellen eine seltene und heterogene Gruppe von Erkrankungen dar, welche meist durch eine leichte bis mittelschwere Blutungsneigung auffallen. Typische Blutungssymptome sind Hämatomneigung, Epistaxis, Menorrhagien sowie Schleimhaut- und perioperative Blutungen. Die Durchführung der Thrombozytenfunktionsdiagnostik bei Kindern wird erschwert durch die altersabhängig begrenzte Blutprobenmenge, schwierige Venenverhältnisse und das Fehlen von Referenzbereichen für Kinder unterschiedlichen Alters. Aufgrund der meist komplizierten und zeitaufwendigen Tests ist die Thrombozytendiagnostik auf spezialisierte Zentren begrenzt. Mit hoher Wahrscheinlichkeit wird eine relevante Anzahl von Kindern mit nichtdiagnostizierten bzw. unkorrekt klassifizierten, klinisch relevanten Thrombozytopathien übersehen. Die Erhebung der Blutungsanamnese und die Bewertung der Blutungssymptome sind erforderlich für eine stufenweise erfolgreiche Gerinnungsdiagnostik. Vor Durchführung einer Thrombozytenfunktionsdiagnostik sollten das Vorliegen einer Thrombozytopenie, einer von-Willebrand-Erkrankung und sekundärer Gerinnungsstörungen ausgeschlossen werden. Die Lichttransmissionsaggregometrie gilt noch immer als Standardmethode für die Beurteilung der Thrombozytenfunktion. Nach Möglichkeit sollte stets versucht werden, den vorliegenden spezifischen Thrombozytenfunktionsdefekt zu klassifizieren, da dies für eine adäquate Behandlung und eine gezielte genetische Beratung notwendig ist. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Si le phénomène bullaire reste le primum movens à l’origine de l’obstruction vasculaire lors de la décompression, les plaquettes sanguines jouent un rôle déterminant dans la physiopathologie de l’accident de décompression (ADD). L’objectif de ces travaux était d’étudier les mécanismes à l’origine de cette agrégation plaquettaire induit par le phénomène bullaire. La première étape a permis de valider un modèle animal d’ADD chez le rat et de confirmer que le degré d’agrégation constituait un indice fiable de sévérité de l’ADD. La seconde a eu pour but d’étudier différents marqueurs spécifiques de l’activation plaquettaire. La troisième étape a étudié les effets de différents anti-agrégants plaquettaires administrés avant l’exposition. Les résultats confirment que le clopidogrel, un inhibiteur spécifique des récepteurs à l’ADP, a un effet protecteur sur le risque de survenue et la sévérité des ADD en réduisant l’importance de l’agrégation plaquettaire. Ces résultats sont en faveur d’un mécanisme d’agrégation ADP-dépendant conséquence des interactions entre les plaquettes et les bulles circulantes. La génération de thrombine, un autre puissant agoniste plaquettaire, interviendrait dans la genèse d’un état pro-thrombotique loco-régional conséquence des lésions induites par le passage des bulles au contact de l’endothélium vasculaire. Enfin, les résultats montrent le rôle joué par les micro-particules dans la diffusion à distance de cet état pro-thrombotique. Des études à venir devront confirmer l’intérêt d’une utilisation du clopidogrel dans le traitement de l’ADD ainsi que le rôle de l’oxyde nitric, de la prostacycline PGI2 et du shear-stress dans l’effet protecteur des vibrations et de l’exercice physique sur le risque de survenue de l’ADD en réduisant l’agrégation plaquettaire et en optimisant la cinétique d’élimination des bulles circulantes. / If bubble-induced mechanical obstruction of vessels is the central event during decompression, blood platelets play a key role in the pathogenesis of decompression sickness (DCS). But bubble-induced platelet aggregation (BIPA) mechanisms are unknown. In a previous study, we highlighted a post-dive decrease in platelet count in divers. The aims of this study was therefore to validate an experimental model of DCD in rat to clarify relationship between blood platelet and bubble formation, investigate platelet activation by measuring different platelet markers, and to study the effects of different antithrombotic pre-treatments. First, the results clearly indicate that clopidogrel, a powerful ADP-inhibitor, has a protective effect on decompression risk improving significantly DCS outcome and reducing DCS severity. The results point to the predominant involvement of the ADP release in the BIPA mechanism. Second, we showed the participation of thrombin generation, a powerful platelet agonist, in the thrombotic event. If local BIPA seems to be the direct consequence of bubble-blood component interactions in vascular bed, the regional thrombotic event could be the consequence of bubble-induced vessel wall injury with a key role played by micro-particles in the general extend. Further human studies should be aimed at demonstrating that clopidogrel can offer a benefit as an adjuvant in DCS treatment and the role played by nitric oxyd and prostacyclin in the protective effect of vibrations and physical exercise on DCS risk reducing bubble-induced platelet aggregation