ADAMTS13 resistance to protease inhibition / Background: ADAMTS13 is a metalloprotease that regulates the delicate balance between VWF multimeric length and its platelet capturing capacity. Unlike other ADAMTS and coagulation proteases, ADAMTS13 exhibits a prolonged half-life of several days as an active protease, suggesting that it is protected from inhibitors of metalloproteases in blood. Here, we investigate the mechanism by which ADAMTS13 is resistant to protease inhibition. Methods: C-terminal domain truncations of ADAMTS13 (MDTCS and MD) and chimeras with ADAMTS5 (MD13/TCS5, M13/DTCS5, MD5/TCS13, and MD5(TCS-CUB13)) were generated. Metalloprotease domain segments from ADAMTS5 were swapped into MDTCS13 corresponding to the gatekeeper triad (R193, D217, and D252) (MDTCS-G), the variable loop (G236-S263) (MDTCS-V5), and the calcium-binding loop (R180-R193) (MDTCS-C5). MDTCS-GVC5 was generated to study these features simultaneously. Alpha 2-macrogloublin (A2M), tissue inhibitors of metalloproteinases (TIMPs), and small molecule inhibitor (Marimastat) were used as inhibitors, and tested using FRETS-VWF73 and Western blot. Results: MDTCS, MD, MD13/TCS5, M13/DTCS5, MDTCS-G, MDTCS-V5, and MDTCS-C5 constructs were resistant to all inhibitors, whereas MD5/TCS13 was inhibited. The presence of the closed conformation attenuated MD5(TCS-CUB13) proteolysis by 50-fold, while displaying a slower rate of inhibition compared to MD5/TCS13. We report the kinetic parameters of the unique features of the metalloprotease domain (the gatekeeper triad, the variable loop, and the calcium-binding loop). Moreover, simultaneously swapping these features sensitized MDTCS-GVC5 to Marimastat. Conclusion: Our findings reveal that the closed conformation confers global latency, while the metalloprotease domain confers local latency of ADAMTS13. The local latency is maintained by the flexibility of the variable loop and the calcium-binding loop, which fold across the active site cleft to restrict inhibitor and substrate access. Extensive engagement of exosites by VWF can readily displace these loops, thereby activating ADAMTS13 from its latent form. Altogether, we present novel insight into the mechanism by which ADAMTS13 is resistant to protease inhibition. / Thesis / Doctor of Philosophy (PhD) / Hemostasis is the body’s natural process to prevent bleeding and maintain blood flow. The ability of a blood protein, called VWF, to stop bleeding upon injury is regulated by the protein ADAMTS13. ADAMTS13 circulates in the blood for days, but its function cannot be stopped by inhibitors. Here, we investigate the mechanism by which ADAMTS13 is resistant to inhibition. We found that several structures of ADAMTS13, called domains and loops, protect it from inhibitors. Folding of the distal domains to the centre of ADAMTS13 partially protected ADAMTS13 from inhibitors. Further investigation revealed that two flexible loops close to the active site of ADAMTS13 were primarily responsible for protecting ADAMTS13 from inhibitors. We suggest that the flexibility of these loops guard against inhibition by folding across the active site. These results are important because advances have been made to use ADAMTS13 therapeutically in many clotting illnesses, such as strokes.

No description available.

The binding between the 45 kDa N-terminal domain of the a subunit of the GPIb-IX-V complex (GPIbαN) on the platelet membrane and the A1 domain of von Willebrand factor (VWF-A1), a multimeric protein circulating in the plasma, plays a key role in platelet adhesion and thrombus initiation at sites of cut-injury and atherosclerotic plaque rupture where blood vessels are subjected to high haemodynamic shear. A fundamental yet unresolved issue is how haemodynamic force upregulates this interaction (binding kinetics) and how a mechanical stimulus is translated into a biochemical signal (mechanotransduction). In order to address above issues, we setup a new biomembrane force probe (BFP) with the drifting reduction, temperature control and concurrent fluorescence imaging. My research findings are summarized into three aims: 1. VWF regions surrounding A1 hinder A1-GPIbα interaction at zero force, which is relieved by increasing force that stabilizes the interaction, giving rise to a VWF-GPIbα catch bond. 2. Three transport-related physical factors: receptor-ligand separation distance, Brownian motion and diffusivity govern the VWF-GPIbα association. 3. Mechanical force and structural variation regulate platelet signaling via the engagement duration of GPIbα mechanosensor. My thesis study advances our understanding of the biophysical and structural basis of how the VWF activation, its interaction with GPIbα and signal transduction are regulated by force when platelets' haemostatic functions are most needed.

Von Willebrand factor (VWF) is a plasma glycoprotein that can bind collagen at a wound site as well as circulating platelets. VWF forms high molecular weight multimers (>20,000 kDa). VWF can also form VWF strings that appear to be attached to the endothelial surface and are capable of binding platelets. These strings are only observed in vitro and in vivo in the absence of the VWF-cleaving protease ADAMTS13. Deficiency in ADAMTS13 results in thrombotic thrombocytopenic purpura (TTP), a clotting disorder characterized by thrombocytopenia, microangiopathic hemolytic anemia, renal dysfunction, neurological dysfunction and fever. Patients suffering from TTP demonstrate VWF-and platelet-rich thrombi in the microvasculature of numerous organ systems, but most notably in the kidneys, heart, and brain. While VWF strings have not been directly connected to TTP, their presence in vivo was only identified with the ADAMTS13 knockout mouse (a model of TTP), suggesting a possible relationship. Recently we identified glycerol as an agent, similar to histamine, that triggers the formation of VWF strings in vitro. We found that glycerol and histamine trigger TTP in an ADAMTS13-deficient mouse model. In addition, we determined conditions in vitro that promote the formation of dense VWF networks. These networks of VWF can be greater than 70 μm thick and appear to be able to form fibers as long as several millimeters in length. These networks have not been previously identified and may underlie a possible mechanism by which VWF-rich thrombi form in TTP. These networks were formed solely from cultured endothelial cells, leading us to believe that endothelial cells alone are capable of producing more VWF than perhaps previously appreciated. These data suggest that secretion of VWF from the endothelium may play an important role in the pathophysiology of TTP.

Obesity is increasing globally and related to major changes in lifestyle. This increase is associated with an increased risk of cardiovascular disease (CVD). Knowledge about adipose tissue as a metabolic-endocrine organ has increased during the last few decades. Adipose tissue produces a number of proteins with increased body weight, many of which are important for food intake and satiety, insulin sensitivity, and vessel integrity, and aberrations have been related to atherosclerosis. Notably, the risk for developing CVD over the course of a lifetime differs between men and women. In Northern Sweden, men have a higher risk for myocardial infarction (MI). However, the incidence is declining in men but not in women. These sex differences could be due to functional and anatomical differences in the fat mass and its functions. The primary aim of this thesis was to evaluate associations between the adipocyte-derived hormones leptin and adiponectin, and fibrinolysis and other variables associated with the metabolic syndrome, and particularly whether these associations differ between men and women. Another aim was to evaluate these associations during physical exercise and pharmacological intervention (i.e. enalapril). Finally, whether leptin and adiponectin predict a first MI or sudden cardiac death with putative sex differences was also investigated. The first study used a cross-sectional design and included 72 men and women  recruited from the WHO MONICA project. We found pronounced sex differences in the associations with fibrinolytic variables. Leptin was associated with fibrinolytic factors in men, whereas insulin resistance was strongly associated with all fibrinolytic factors in women. The second study was an experimental observational study with 20 men exposed to strenuous physical exercise. During exercise, leptin levels decreased and adiponectin levels increased, and both were strongly associated with an improved fibrinolytic capacity measured as decreased PAI-1 activity. Changes in insulin sensitivity were not associated with changing adiponectin levels. The third study was a randomised, double-blind, single centre clinical trial including 46 men and 37 women who had an earlier MI. The study duration was one year, and participating subjects were randomised to either placebo or ACE inhibitor (i.e. enalapril). Circulating leptin levels were not associated with enalapril treatment. During the one-year study, changes in leptin levels were associated with changes in circulating levels of tPA mass, PAI-1 mass, and tPA-PAI complex in men, but not vWF. These associations were found in all men and men on placebo treatment. In women on enalapril treatment there was an association between changes in leptin and changes in vWF. In the fourth study, the impact of leptin, adiponectin, and their ratio on future MI risk or sudden cardiac death was tested in a prospective nested casecontrol study within the framework of the WHO MONICA, Västerbotten Intervention Project (VIP), and Västerbotten  Mammary Screening Program (MSP). A total 564 cases (first-ever MI or sudden cardiac death) and 1082 matched controls were selected. High leptin, low adiponectin, and a high leptin/adiponectin ratio independently predicted a first-ever MI, possibly with higher risk in men in regards to leptin. The association was found for non-fatal cases with ST-elevation MI. Subjects with low adiponectin levels had their MI earlier than those with high levels. In conclusion, the adipocyte-derived hormones leptin and adiponectin are related to the development of CVD with a sex difference, and fibrinolytic mechanisms could be possible contributors to CVD risk.

Thrombosis is initiated by platelets and leads to cardio-, cerebro-, and peripheral vascular disease, the leading causes of morbidity and mortality in the western world. Antiplatelet drugs have improved clinical outcomes for thrombosis patients. However, their expanded use is limited by hemorrhage at high concentrations and sub-therapeutic activity at lower doses. Thus, development of new antiplatelet agents with improved safety and efficacy is a medical priority. VWF is a multimeric plasma glycoprotein that plays a critical role in platelet-mediated thrombus formation and presents an attractive target for antiplatelet therapy. To this end, I have isolated and characterized aptamer molecules that bind to VWF with high affinity and have shown that some of these aptamer molecules could inhibit platelet activation/aggregation in vitro and in vivo. Furthermore, I designed antidote molecules that can reverse the effects of the aptamer molecules, restoring platelet function quickly and effectively. This project has yielded the first antidote controlled antiplatelet agent and may lead to significant improvements in thrombosis therapy. Thrombin is a plasma protein that plays a critical role in thrombosis. Currently, available antithrombin agents are efficacious in preventing coagulation but do not significantly affect platelet activation and aggregation, both essential components of thrombus formation. Therefore, I tested two aptamer molecules that bind to mutually exclusive exosites on thrombin and, when used together, synergistically inhibit both coagulation and platelet activation. I demonstrated that this method could potentially lead to the development of effective antithrombotic therapies. With an ever-increasing number of people taking multiple medications, the need to safely administer drugs and limit unintended side effects has never been greater. Antidote control remains the most direct means to counteract acute side effects of drugs but unfortunately it has been challenging and cost prohibitive to generate antidotes for most therapeutic agents. Therefore, I described the development of a set of antidote molecules that are capable of counteracting the effects of an entire class of therapeutic agents, i.e. aptamers, including those that I generated against VWF. I demonstrated that protein and polymer-based molecules that capture oligonucleotides can reverse the activity of aptamers in vitro and in vivo. / Dissertation

Arterial thrombus forms from the capture and accumulation of circulating platelets on a stenosis. As the thrombus grows, the lumen becomes further stenotic producing very high shear rates as the blood velocities increase through the narrowed cross-section. This study explores the molecular binding conditions that may occur under these pathologic shear conditions where circulating platelets must adhere quickly and with strong bonds. Platelets binding in an arterial stenosis of >75% are subject to drag forces exceeding 10,000 pN. This force can be balanced by 100 simultaneous GPIb-vWFA1 bonds of 100 pN each. The number and density of GPIb on platelets is sufficiently high; however, platelet capture under high shear would require the density of A1 receptors to be increased to over 416 per square micron. A computational model is used to determine platelet capture as a function of shear rate, surface receptor density, surface contact and kinetic binding rate. A1 density could be increased by a combination of vWF events of: i) plasma vWF attach to the thrombus surface and elongate under shear; ii) the elongated vWF strands create a net with 3-D pockets; and iii) additional vWF is released from mural platelets by activation under shear. With all three events, A1 density matches the existing high GPIbα densities to provide sufficient multivalency for capture at 100,000 s-1 with greater than 170 bonds per platelet. If the on-rate is greater than 108 M-1s-1, then a platelet could be captured within 15 microseconds, the amount of time available to form bonds before the platelet is swept away. This mechanism of platelet capture allows for the rapid platelet accumulation in atherothombosis seen clinically and in high shear experiments.