New findings on venous thrombogenesis

Venous thrombosis (VT) is the third most common cause of cardiovascular death worldwide. Complications from VT and pulmonary embolism are the leading cause of lost disability-adjusted life years. Risks include genetic (e.g., non-O blood group, activated protein C resistance, hyperprothrombinemia) and acquired (e.g., age, surgery, cancer, pregnancy, immobilisation, female hormone use) factors. Pathophysiologic mechanisms that promote VT are incompletely understood, but involve abnormalities in blood coagulability, vessel function, and flow (so-called Virchow’s Triad). Epidemiologic studies of humans, animal models, and biochemical and biophysical investigations have revealed contributions from extrinsic, intrinsic, and common pathways of coagulation, endothelial cells, leukocytes, red blood cells, platelets, cell-derived microvesicles, stasis-induced changes in vascular cells, and blood rheology. Knowledge of these mechanisms may yield new therapeutic targets. Characterisation of mechanisms that mediate VT formation and stability, particularly in aging, are needed to advance understanding of VT

Systems biology of platelet-vessel wall interactions

Platelets are small, anucleated cells that participate in primary hemostasis by forming a hemostatic plug at the site of a blood vessel's breach, preventing blood loss. However, hemostatic events can lead to excessive thrombosis, resulting in life-threatening strokes, emboli, or infarction. Development of multi-scale models coupling processes at several scales and running predictive model simulations on powerful computer clusters can help interdisciplinary groups of researchers to suggest and test new patient-specific treatment strategies

A Systems Approach to Hemostasis: How the Feedback Between Thrombus Structure and Molecular Transport Regulates the Hemostatic Response

After vascular injury numerous chemical signals are released to induce platelet activation, coagulation, and post-hemostatic events. This thesis aims to investigate the interplay between thrombus structure and the spatiotemporal distribution and transport of biologically relevant solutes, and how this impacts thrombus formation in vivo. Using intravital microscopy we have previously described a characteristic architecture of thrombi formed in vivo. The architecture consists of a core of highly-activated and tightly packed platelets covered by a loose shell of less activated platelets. Initially, we developed a novel platelet-targeted sensor capable of reporting on thrombin activity, a potent platelet agonist, within thrombi formed ex vivo or in vivo. We found that thrombin activity was high in the core region, but restricted from the shell. We then designed another sensor capable of tracking soluble protein transport within thrombi formed in vivo, and found significant retention of soluble proteins within the platelets that would go on to form the core region. Using computational methods we found that the platelet packing density between the platelets restricted the diffusion of proteins within the core region, and allowed for rapid elution of proteins that made it to the shell. To test this in vivo we used mice with a defect in platelet retraction, but not platelet sensitivity to agonists. The mutant mice showed a much faster rate of solute elution using our transport sensor, and we also observed decreased platelet activation and thrombin activity within the thrombus. Next, we extended this model of thrombi as regulators of protein transport by examining how thrombus architecture altered the leakage of plasma proteins into the surrounding tissue. We found that extravascular solute gradients were sensitive to commonly used anti-platelet agents as well as small changes in platelet packing densities. Finally, we developed a new intravital imaging technique to visualize thrombus architecture formation in the mouse femoral artery and vein to extend our observations into the macrocirculation. Together, this thesis proposes a novel mechanism of thrombus regulation, which is dependent upon molecular transport properties shaped by the local hemodynamics and the intrathrombus microenvironment

A Microfluidic Approach For Evaluating Novel Antithrombotic Targets

Microfluidic systems allow precise control of the anticoagulation/pharmacology protocols, defined reactive surfaces, hemodynamic flow and optical imaging routines, and thus are ideal for studies of platelet function and coagulation response. This thesis describes the use of a microfluidic approach to investigate the role of the contact pathway factors XII and XI, platelet-derived polyphosphate, and thiol isomerases in thrombus growth and to evaluate their potential as safer antithrombotic drug targets. The use of low level of corn trypsin inhibitor allowed the study of the contact pathway on collagen/kaolin surfaces with minimally disturbed whole blood sample and we demonstrated the sensitivity of this assay to antithrombotic drugs. On collagen/tissue factor surfaces, we found the relative contributions of the extrinsic pathway, the contact pathway, and the thrombin feedback pathway vary with tissue factor surface concentration. Platelet-derived polyphosphate potentiated the thrombin feedback pathway at low tissue factor level but enhanced fibrin fiber structure regardless of tissue factor level. At locations with low tissue factor level, thrombosis may be druggable by contact pathway and polyphosphate inhibition, although thrombolytic susceptibility may benefit from polyphosphate antagonism regardless of tissue factor level. We developed a peptide-based platelet-targeting thiol reduction sensor to visualize thrombus-incorporated thiol reductase activity. Although distribution of thiol reductase activity was shown to be correlated with the level of platelet activation, protein disulfide isomerase inhibition showed a limited effect on platelet aggregation in microfluidic thrombosis assay. We also used the microfluidic system to explore the injury patch size limit for triggering clotting. We observed a full clotting response of platelet deposition, thrombin generation and fibrin polymerization on one of the smallest biological units of a single collagen fiber presenting tissue factor and von Willebrand factor suggesting the lack of physiological injury patch size limit. Finally, we made the first estimation of thrombin flux from growing thrombus under flow using the microfluidic thrombosis assay in combination with enzyme-linked immunosorbent measurement of thrombin-antithrombin complex. We found thrombin is robustly generated within clots by the extrinsic pathway, followed by late-stage factor XIa contributions, with fibrin localizing thrombin via its antithrombin activity as a self-limiting hemostatic mechanism

Model predictions of deformation, embolization and permeability of partially obstructive blood clots under variable shear flow

Thromboembolism, one of the leading causes of morbidity and mortality worldwide, is characterized by formation of obstructive intravascular clots (thrombi) and their mechanical breakage (embolization). A novel two-dimensional multi-phase computational model is introduced that describes active interactions between the main components of the clot, including platelets and fibrin, to study the impact of various physiologically relevant blood shear flow conditions on deformation and embolization of a partially obstructive clot with variable permeability. Simulations provide new insights into mechanisms underlying clot stability and embolization that cannot be studied experimentally at this time. In particular, model simulations, calibrated using experimental intravital imaging of an established arteriolar clot, show that flow-induced changes in size, shape and internal structure of the clot are largely determined by two shear-dependent mechanisms: reversible attachment of platelets to the exterior of the clot and removal of large clot pieces. Model simulations predict that blood clots with higher permeability are more prone to embolization with enhanced disintegration under increasing shear rate. In contrast, less permeable clots are more resistant to rupture due to shear rate-dependent clot stiffening originating from enhanced platelet adhesion and aggregation. These results can be used in future to predict risk of thromboembolism based on the data about composition, permeability and deformability of a clot under specific local haemodynamic conditions

Platelets mediate lymphovenous hemostasis to maintain blood-lymphatic separation throughout life

Mammals transport blood through a high-pressure, closed vascular network and lymph through a low-pressure, open vascular network. These vascular networks connect at the lymphovenous (LV) junction, where lymph drains into blood and an LV valve (LVV) prevents backflow of blood into lymphatic vessels. Here we describe an essential role for platelets in preventing blood from entering the lymphatic system at the LV junction. Loss of CLEC2, a receptor that activates platelets in response to lymphatic endothelial cells, resulted in backfilling of the lymphatic network with blood from the thoracic duct (TD) in both neonatal and mature mice. Fibrin-containing platelet thrombi were observed at the LVV and in the terminal TD in wild-type mice, but not Clec2-deficient mice. Analysis of mice lacking LVVs or lymphatic valves revealed that platelet-mediated thrombus formation limits LV backflow under conditions of impaired valve function. Examination of mice lacking integrin-mediated platelet aggregation indicated that platelet aggregation stabilizes thrombi that form in the lymphatic vascular environment to prevent retrograde blood flow. Collectively, these studies unveil a newly recognized form of hemostasis that functions with the LVV to safeguard the lymphatic vascular network throughout life