Neutrophils promote venular thrombosis by shaping the rheological environment for platelet aggregation

In advanced inflammatory disease, microvascular thrombosis leads to the interruption of blood supply and provokes ischemic tissue injury. Recently, intravascularly adherent leukocytes have been reported to shape the blood flow in their immediate vascular environment. Whether these rheological effects are relevant for microvascular thrombogenesis remains elusive. Employing multi-channel in vivo microscopy, analyses in microfluidic devices, and computational modeling, we identified a previously unanticipated role of leukocytes for microvascular clot formation in inflamed tissue. For this purpose, neutrophils adhere at distinct sites in the microvasculature where these immune cells effectively promote thrombosis by shaping the rheological environment for platelet aggregation. In contrast to larger (lower-shear) vessels, this process in high-shear microvessels does not require fibrin generation or extracellular trap formation, but involves GPIb alpha-vWF and CD40-CD40L-dependent platelet interactions. Conversely, interference with these cellular interactions substantially compromises microvascular clotting. Thus, leukocytes shape the rheological environment in the inflamed venular microvasculature for platelet aggregation thereby effectively promoting the formation of blood clots. Targeting this specific crosstalk between the immune system and the hemostatic system might be instrumental for the prevention and treatment of microvascular thromboembolic pathologies, which are inaccessible to invasive revascularization strategies

A multilevel study of platelet activation at biomaterial surface: from evaluatinhg to controlling hemocompatibility

CVD are the major cause of death world-wide. Their management relies on artificial materials in implants and external devices. They cause thrombotic and inflammatory complications and require therapies that are costly and dangerous for the patient. The research into blood-material interactions failed to find hemocompatible materials, an adequate in vitro test for evaluating hemocompatibility, or appreciate the complexity of the blood-biomaterial interactions

Crosstalk between melanoma cells and the blood-brain barrier: Impact on coagulation and brain metastasis to identify new anti-metastatic targets

Brain metastasis is associated with a high risk for venous thromboembolism (VTE), which determines the prognosis of the patient. We postulated that von Willebrand factor (VWF), a procoagulant glycoprotein stored in endothelial cells (ECs) and platelets, contributes to VTE and promotes metastasis in the brain. Previously, it was shown that EC activation and the subsequent formation of luminal VWF fibers mediate the recruitment of platelets promoting tumor-associated vessel occlusion and pulmonary metastasis. However, little is known about the function of VWF within the specialized vascular bed of the blood-brain barrier. The aim of this project is to determine the contribution of VWF in the pathophysiology of brain metastasis. Using in vitro approaches significant lower levels of VWF were detected in primary human microvascular brain ECs compared to macrovascular human umbilical vein ECs. This was reflected by a restricted release of VWF and low numbers of luminal VWF fibers upon tumor cell-induced brain EC activation. In line, brain microvessels of wild type mice showed low amounts of stored VWF and few VWF-platelet aggregates. However, immunofluorescence analyses of brain tissue from patients with brain metastasis revealed a strong formation of luminal VWF-platelet aggregates mediating vessel occlusions. These findings were confirmed in the ret transgenic mouse model, which develops spontaneous melanoma with metastasis in the brain: metastatic ret mouse brains showed a strong formation of intravascular VWF-platelet aggregates. Importantly, this phenomenon was already observed in brains of ret transgenic mice without visible cerebral metastasis, suggesting that luminal VWF fibers are involved in initial steps of the brain metastatic cascade. High resolution microscopy techniques revealed the contribution of activated platelets in the formation of luminal VWF networks in the brain. Consistent with this, aggregometry assays and in vitro microfluidic model of brain microcirculation showed that the lack of VWF in platelets (VWF-/-) resulted in a reduced platelet aggregation. Additionally, impedance measurements and transmigration assays demonstrated that blocking platelet activation with the low molecular weight heparin Tinzaparin reduces the impact of platelet-mediated endothelium disruption and the transmigration of tumor cells. Furthermore, systemic anticoagulation using Tinzaparin reduced platelet accumulation and VWF networks in ret mouse brains. The impact of anticoagulation on brain metastasis formation was examined after intracardiac injection of melanoma cells in mice treated with Tinzaparin. Quantification of brain metastasis showed that Tinzaparin attenuates the metastatic load compared to non-treated control mice. In summary, our findings provide new insights into the mechanisms by which platelet-derived VWF promotes cerebral thrombosis and identifies platelet activation as a promising therapeutic target for the prevention of brain metastases

Characterization of the structural-functional impact of heterozygous missense mutations in genes of the blood coagulation factor XIII that cause mild Factor XIII deficiency

The coagulation Factor XIII is a key player in hemostasis that is responsible for the last step of the coagulation cascade in which it covalently cross-links preformed fibrin clots to make them resistant to premature fibrinolysis. The plasma circulating FXIII zymogen is a heterotetrameric complex comprising two catalytic A (FXIIIA2) and two protective carrier B-Subunits (FXIIIB2) which are synthesized and secreted into the plasma as homo-dimers from various cell types like monocytes/ macrophages, megakaryocytes, and platelets for the A subunit and hepatocytes for the B subunit. Deficiency of FXIII results in a bleeding predisposition for the individual carrying it. This deficiency can have congenital or acquired origins. The inherited form of FXIII deficiency can be classified into two types based on severity of symptoms: severe and mild FXIII deficiency. While the homozygous inherited form of this deficiency caused by F13A1 (OMIM #613225) (FXIIIA subunit) or FXIIIB (OMIM #613235) (FXIIIB subunit) gene mutations is rare (1 in 2-4 million), the milder heterozygous form is more frequent. Only recently, focus has shifted to the mild/heterozygous form of this deficiency that is associated with mild or even an asymptomatic phenotype (unless the affected individual is exposed to some kind of a trauma for e.g. peri-operative settings, accident etc.). Recent investigations from our group in the past five years have shown that inherited mild heterozygous deficiency does have clinical relevance. Identification of heterozygous FXIII deficient patients and extended causality determining research on the related mutations is crucial since the risk of provoked bleeding events (surgery, tooth extraction, trauma) in heterozygous patients can be minimized through early detection. In the last five years, our group has reported 23 mutations from patients with mild FXIII deficiency. Sixteen of these mutations were identified in the F13A1 and seven in the F13B gene. In the present study we have performed a comprehensive investigation on the causality of these reported missense mutations using parallel in silico and in vitro approaches to structurally and functionally characterize their underlying pathophysiology. The in vitro methods have been complemented by in silico strategies in which modeling of protein subunits/domains/mutations and simulation/docking based approaches have been applied to explain the in vitro findings. Our analysis shows that these mutations can act on different aspects of FXIII activation and regulation based on the structure functional impact of the particular mutation

Prevention and treatment of hemostatic complications in liver disease

This thesis on the prevention and treatment of hemostatic complications in liver disease starts with a detailed study on the hemostasis in cirrhosis. Our in-depth hemostatic profiling of primary, secondary, and tertiary hemostasis in a group of patients with CPT A/B cirrhosis showed no large differences between etiologies, and was consistent with a general hypercoagulable profile in patients with mild cirrhosis. These results suggest that patients with cirrhosis have an increased risk of thrombosis, irrespective of their etiology.Whether this translates to a similar effect of anticoagulants was tested in vitro in patients with cirrhosis due to ASH or NASH. Low-molecular-weight-heparin, dabigatran and apixaban were tested and showed no differences on a hemostatic level between groups.For the in vivo testing of anticoagulants we studied the effect of a therapeutic dose of edoxaban in patients and controls. Edoxaban strongly effects the hemostatic activity in patients, but not as much as in controls. Whether this difference translates into a higher risk of thrombosis and necessitates dose-adjustments in patients with cirrhosis should be further assessed.In vitro testing of commonly used anticoagulants in patients undergoing hepato-pancreatico-bilairy surgery showed a profoundly altered efficacy as compared to healthy controls, which may have implications for anticoagulant dosing in the early postoperative period. In the correction of perioperative bleeding complications, prothrombin complex concentrate (PCC) appear much more potent than fresh frozen plasma or recombinant factor VIIa, and PCCs may require conservative dosing and caution in use in patients undergoing HPB surgery

Microfluidic Approaches to Thrombosis and Hemostasis: Towards a Patient-Specific Test of Antiplatelet Therapeutics and the Assessment of Coagulopathy in Hemophilic and Trauma Patients

Current in vitro or ex vivo models of hemostasis and thrombosis fail to recapitulate the hemodynamic conditions and biorheologic phenomena found throughout the vasculature. Microfluidic technology enables physiologic hemodynamics for the study of platelet deposition and coagulation using minimum volumes of human whole blood. This dissertation describes the application of microfluidic assays, the manipulation of surface-patterned procoagulant and sub-endothelial proteins, anti-coagulation, and flow conditions to investigate platelet function and coagulation under flow. First, we demonstrate a novel method to assess the in vivo or in vitro therapeutic efficacy of anti-platelet therapies on platelet aggregates adhering to collagen type I surfaces. We phenotyped individual healthy donor platelet function responses to in vivo or in vitro aspirin, a common antiplatelet therapy over collagen type I surfaces at venous shear rates. Utilizing the same flow assay, we also characterized mechanism-based resistance to aspirin conferred by non-steroidal anti-inflammatory drugs. Furthermore, we have also developed a new model to assess the intrinsic pathway of coagulation under flow on collagen type I surfaces and investigated the role of the intrinsic pathway in recombinant coagulation factor VIIa (rFVIIa) therapeutic efficacy. We then extended this mechanistic investigation of rFVIIa to flow assays where clotting is initiated by collagen and immobilized lipidated tissue factor to evaluate the role of the intrinsic tenase in conjunction with exogenous rFVIIa when surface-triggered extrinsic pathway is present. Finally, we continued to assess coagulopathic patients by first mimicking resuscitation-driven hemodilution, hyperfibrinolysis, and plasmin-inhibitor therapy under flow. We then evaluated downregulation of platelet function in whole blood from trauma patients during the acute phase of trauma-induced coagulopathy. The development of microfluidics, microfabrication, and its applications in hemostasis and thrombosis is essential in advancing our knowledge of clinical and pathological disorders such as myocardial infracts, hemophilia, and deep vein thrombosis. Beyond this work, microfluidic platforms in hemostasis and thrombosis can potentially be used as drug screening platforms for antiplatelet or clotting factor therapies, or a point of care diagnostic test for bleeding and pin-pointing the therapeutic index of novel biopharmaceutics

Molecular mechanisms impairing biosynthesis and function of hemostatic proteins

The gene mutations leading to hemorrhagic disorders provide peculiar models to elucidate molecular mechanisms underlying protein biosynthesis, and the relationship between the structure and function. The research activity has been focused on the molecular defects leading to severe deficiency of Factor IX (FIX), a serine protease with a key role in the intrinsic pathway of blood coagulation, and of von Willebrand factor (VWF), a large multimeric protein essential for the primary hemostasis. In particular, I investigated mechanisms due to three different molecular defects such as nonsense and missense mutations in F9 gene, associated with type I hemophilia B, and an in-frame deletion in the VWF gene, displaying a dominant-negative effect. Results from investigations in patients’ plasma and the expression of nonsense FIX variants in eukaryotic cells led to the demonstration of trace levels of full-length FIX molecules even in the presence of nonsense mutations through a mechanism of ribosome read-through. The efficiency was dependent of the specific nonsense mutation and on it sequence context. Moreover, I investigated the susceptibility of a panel of nonsense FIX mutations to the induction of readthrough by aminoglycosides. The data suggested a direct relationship between the spontaneous and the drug-induced readthrough. Overall data indicated that not all nonsense mutations can be considered truly “null-mutations”, a finding that have pathophysiological implications. The severe p.Tyr450Cys mutation in the carboxyl-terminal region of coagulation FIX was chosen as model to study the interplay between impaired protein biosynthesis and/or function caused by missense mutations in relation to specific protein regions, which has been poorly investigated. Results from the expression of a panel of recombinant variants demonstrated the key role of the tyrosine phenyl group for both FIX secretion and coagulant activity. Comparison among highly homologous coagulation serine proteases indicate that additive or compensatory pleiotropic effects on secretion and function by carboxyl-terminus mutations produce life-threatening or mild phenotypes in the presence of similarly reduced protein amounts. Finally I contributed to the characterization of the dominant inheritance in VWD, due to two essential process in VWF dimerization and multimerization VWD can express dominant-negative features. Previous study characterized and demonstrate d the modulation of this dominant effect. In our study we reproduced in vivo the effect of dominance and through a creation of an artificial mutation, we demonstrated in vitro an in vivo the key role of interaction between wild type and mutant protein monomers during dimerization and or multimerization, We believe that our finding have general implication for the dominant forms of VWD, the most frequent inherited bleeding disorders in humans