Molecular Mechanism in the Alteration of Hemostasis

Hemostasis is a finely tuned physiological process that, through the concerted action of several blood cells and proteins, maintains the integrity of the vascular system. This stepwise process begins after a vessel wall injury and includes: an initial vasospasm, a platelet plug formation (primary hemostasis), an assembly and activation of the coagulation factors that results in fibrin deposition at the site of injury (secondary hemostasis), and a final dissolution of the fibrin clot that restores the blood vessel patency (fibrinolysis) (Chapter 1). Alterations affecting one or more of these delicate processes lead to a large number of pathological manifestations, commonly referred to as cardiovascular diseases (CVD). Nowadays, CVD are the major cause of mortality and morbidity worldwide. Despite the social and economic burden of CVD, the currently available pharmaceutical repertoire is relatively limited to a few classes of molecules (heparins, platelet antiaggregants, vitamin-K antagonists, direct thrombin inhibitors) which, however, display important side effects and need to be employed with careful dose adjustments. These difficulties stem primarily from: i) the intrinsically complex nature of the procoagulant and anticoagulant biochemical mechanisms leading to physiological hemostasis, which renders external intervention very risky and unpredictable; ii) the inadequate knowledge of the biochemical mechanisms linking blood coagulation to other vital physio-pathological processes. The general aim of this Ph.D. project was to investigate some of the molecular mechanisms underlying hemostatic disorders. To address this relevant question, we proceeded by studying selected pathologies for which association with hemostatic complications has either been long-established (i.e., Antiphospholipid Syndrome (APS), infectious diseases) or has just been hypothesized (Parkinson’s disease (PD), Transthyretin-related Amyloidosis (ATTR)), focusing our attention on the physio-pathological proteins involved in the onset of these disorders. In a first stage, our attention was focused on the study of novel interactions between α-thrombin (αT), the key enzyme of the coagulation cascade, with other plasma proteins (i.e., β2-glycoprotein-I, α-synuclein). In a second stage, we investigated an alternative mechanism of activation of prothrombin, the precursor of αT, by a bacterial protease (subtilisin from B. subtilis). Finally, some selected proteases were tested against human transthyretin, whose proteolyzed form is a key factor in the onset of ATTR. In its traditional pathway, blood coagulation culminates with the FXa-mediated conversion of prothrombin zymogen into active αT, through the formation of the prothrombinase complex on the platelet surface. Mature αT is a 36.7 kDa serine protease with a chymotrypsin-like fold. αT plays a pivotal role in blood coagulation, being able to exert both procoagulant (platelets aggregation, fibrin generation) and anticoagulant (protein C activation) functions. The equilibrium between such different activities is regulated by the interaction of αT with other proteins through its active site and two positively charged regions, called exosites (exosite I and exosite II), which flank the catalytic cleft. In addition, αT is a multifunctional protease that, beyond blood coagulation, plays important roles also in other physiological processes such as inflammation, innate immune system, and nervous systems. In Chapter 2 we mapped the interaction between αT and β2-Glycoprotein I (β2GpI). β2GpI is a heavily glycosylated 45 kDa protein that resides in human plasma at a physiological concentration of 4 µM (0.25 mg/ml). Since the early 90's, β2GpI has been identified as the major autoantigen in the antiphospholipid syndrome (APS), a severe autoimmune disease clinically characterized by hemostatic alterations such as venous and arterial thrombosis, fetal loss and thrombocytopenia. Despite its involvement in the pathogenesis of APS, the physiological roles of β2GpI remain unclear and both pro- and anti-coagulant functions have been reported for this protein. In a recent work, we have shown that β2GpI selectively inhibits the procoagulant functions of human α-thrombin (i.e. prolongs fibrin clotting time, tc, and inhibits α-thrombin-induced platelets aggregation) without affecting the unique anticoagulant activity of the protease (i.e. the proteolytic generation of the anticoagulant protein C). Here, combining molecular modeling with biochemical/biophysical techniques, we provided a coherent structural model of αT-β2GpI complex. The model has allowed us to understand at the molecular level our previous in vitro results. In particular, our findings suggested that β2GpI may function as an anticoagulant protein, acting as a scavenger of αT for the binding to GpIbα receptor, thus impairing platelets aggregation while enabling normal cleavage of fibrinogen and protein C. Chapter 3 was dedicated to the role of bacterial proteases in inducing blood coagulation by direct proteolytic activation of prothrombin. This knowledge gap is particularly concerning, as bacterial infections are frequently complicated by severe coagulopathies, and, in about 35% of sepsis cases, by disseminated intravascular coagulopathies (DIC). Here, we show that addition of subtilisin (50 nM–2 µM), a serine protease secreted by the nonpathogenic bacterium Bacillus subtilis, to human plasma induces clotting by proteolytically converting prothrombin into active σPre2, a nicked Pre2 derivative with a single cleaved Ala470–Asn471 bond. Notably, we found that this non-canonical cleavage at Ala470–Asn471 is instrumental for the onset of catalytic activity in σPre2, which was however reduced of about 100-200 fold compared with natural αT. Of note, σPre2 could generate fibrin clots from fibrinogen, either in solution or in blood plasma, and could aggregate human platelets, either isolated or in whole blood. Our findings demonstrate that alternative cleavage of prothrombin by proteases, even by those secreted by non-virulent bacteria such as B. subtilis, can shift the delicate procoagulant-anticoagulant equilibrium toward thrombosis. The study object presented in Chapter 4 is the interplay between αT and α-synuclein (αSyn). αSyn is a small (14.6 kDa) presynaptic protein mainly synthesized in the brain and whose aggregation has been shown to trigger the onset of different neurodegenerative diseases, commonly referred to as synucleinopathies (i.e., Parkinson disease). As for β2GpI, the exact physiological role of αSyn is still elusive. Intriguingly, αSyn is also synthesized by platelets and was found to inhibit the Ca2+-dependent release of procoagulant α-granules after αT stimulation. Moreover, clinical evidences clearly indicate that patients affected by neurodegenerative disorders have lower risks of ischemic attack. The collateral effects of αSyn in the pathogenesis and its localization on platelet surfaces prompted us to investigate a possible role of it in the hemostatic system. Here, we studied the effects of αSyn on fibrin generation and platelet activation. Furthermore, we mapped the interaction sites on αSyn and αT. Briefly, our results indicate that the negatively charged C-terminal tail of αSyn binds to the electropositive exosite-2 of thrombin, thus impairing αT-mediated platelet activation in whole blood. At variance, αSyn does not alter the rate of fibrin generation, resulting only in a minor change in the ensuing fibrin structure. In Chapter 5 we attempted to correlate the onset of systemic transthyretin amyloidosis to an altered activation of blood coagulation. Human transthyretin (hTTR) is an abundant homo-tetrameric plasma protein (0.2 mg/ml) involved in the transport of thyroxine and retinol through the binding to retinol binding protein. Beyond its physiological roles, hTTR is known as an amyloidogenic protein whose aggregation is responsible for several amyloid diseases, including senile systemic amyloidosis (SSA), familial amyloid polyneuropathy (FAP), and familial amyloid cardiomyopathy (FAC). From a mechanistic point of view, the proteolytic cleavage of hTTR represents an important step in fibril formation. In particular, after cleavage around position 50, hTTR C-terminal fragments have been found to aggregate far more efficiently than the full-length hTTR. Nowadays, the protease(s) responsible for this cleavage is yet to be identified although it is predicted to be a serine protease with a trypsin-like fold. Since all coagulation factors are trypsin-like serine proteases, we decided to probe them for the proteolytic cleavage of hTTR. In addition, we also probed some selected bacterial proteases, as well as some digestive apparatus and immune system proteases. hTTR was resistant to all proteases tested except to subtilisin from B. subtilis, which was able to cleave hTTR at pH 7.4, generating in high yields the amyloidogenic fragment hTTR(59-127). Since the hTTR(59-127) fragment was identified in amyloid deposits, these new insights might have relevant implications in hTTR-based amyloidosis