Unique case of autoantibody mediated inactivation of ADAMTS13 in an Indian TTP patient.

A young Indian female visited hospital as a suspected case of thrombotic thrombocytopenic purpura (TTP) with relapsed thrombotic complications with low platelet counts, infarct in middle cerebral artery and thrombi in microvessels. We first confirmed the deficiency of ADAMTS13 metalloprotease in this patient showing improper cleavage of vWF multimers by her plasma unlike her parents and brother. Although patient had very less ADAMTS13 antigen in plasma, but it did not appear to be the cause of deficiency of the enzyme, because her father had similarly low antigen level and he never had prothrombotic complications. While investigating the genetic change in ADAMTS13, we observed four homozygous-SNPs (g.420T>C, g.1342C>G, g.1716G>A and g.2280T>C) in exon 5, 12, 15 and 19 respectively in patient and her father unlike the heterozygous form of same SNPs in mother and brother. Further to investigate the cause of ADAMTS13 deficiency, we observed an elevated level of antibody against ADAMTS13 in patient unlike her father and other family members. Our study therefore provides the molecular approach of diagnosis of TTP in this patient and also highlights the use of such techniques in India. More importantly, study provides the clue of alternate treatment such as immunosuppressant therapy to this patient.

Development of pro-inflammatory phenotype in monocytes after engulfing Hb-activated platelets in hemolytic disorders.

Monocytes and macrophage combat infections and maintain homeostatic balance by engulfing microbes and apoptotic cells, and releasing inflammatory cytokines. Studies have described that these cells develop anti-inflammatory properties upon recycling the free-hemoglobin (Hb) in hemolytic conditions. While investigating the phenotype of monocytes in two hemolytic disorders-paroxysmal nocturnal hemoglobinuria (PNH) and sickle cell disease (SCD), we observed a high number of pro-inflammatory (CD14+CD16hi) monocytes in these patients. We further investigated in vitro the phenotype of these monocytes and found an estimated 55% of CD14+ cells were transformed into the CD14+CD16hi subset after engulfing Hb-activated platelets. The CD14+CD16hi monocytes, which were positive for both intracellular Hb and CD42b (platelet marker), secreted significant amounts of TNF-α and IL-1ß, unlike monocytes treated with only free Hb, which secreted more IL-10. We have shown recently the presence of a high number of Hb-bound hyperactive platelets in patients with both diseases, and further investigated if the monocytes engulfed these activated platelets in vivo. As expected, we found 95% of CD14+CD16hi monocytes with both intracellular Hb and CD42b in both diseases, and they expressed high TNF-α. Furthermore our data showed that these monocytes whether from patients or developed in vitro after treatment with Hb-activated platelets, secreted significant amounts of tissue factor. Besides, these CD14+CD16hi monocytes displayed significantly decreased phagocytosis of E. coli. Our study therefore suggests that this alteration of monocyte phenotype may play a role in the increased propensity to pro-inflammatory/coagulant complications observed in these hemolytic disorders-PNH and SCD.

Hemoglobin interaction with GP1bα induces platelet activation and apoptosis: a novel mechanism associated with intravascular hemolysis.

Intravascular hemolysis increases the risk of hypercoagulation and thrombosis in hemolytic disorders. Our study shows a novel mechanism by which extracellular hemoglobin directly affects platelet activation. The binding of Hb to glycoprotein1bα activates platelets. Lower concentrations of Hb (0.37-3 µM) significantly increase the phosphorylation of signaling adapter proteins, such as Lyn, PI3K, AKT, and ERK, and promote platelet aggregation in vitro. Higher concentrations of Hb (3-6 µM) activate the pro-apoptotic proteins Bak, Bax, cytochrome c, caspase-9 and caspase-3, and increase platelet clot formation. Increased plasma Hb activates platelets and promotes their apoptosis, and plays a crucial role in the pathogenesis of aggregation and development of the procoagulant state in hemolytic disorders. Furthermore, we show that in patients with paroxysmal nocturnal hemoglobinuria, a chronic hemolytic disease characterized by recurrent events of intravascular thrombosis and thromboembolism, it is the elevated plasma Hb or platelet surface bound Hb that positively correlates with platelet activation.

High-mobility group box 1 increases platelet surface P2Y12 and platelet activation in sickle cell disease.

Thrombosis and inflammation are intimately linked and synergistically contribute to the pathogenesis of numerous thromboinflammatory diseases, including sickle cell disease (SCD). While platelets are central to thrombogenesis and inflammation, the molecular mechanisms of crosstalk between the 2 remain elusive. High-mobility group box 1 (HMGB1) regulates inflammation and stimulates platelet activation through Toll-like receptor 4. However, it remains unclear whether HMGB1 modulates other thrombotic agonists to regulate platelet activation. Herein, using human platelets, we demonstrate that HMGB1 significantly enhanced ADP-mediated platelet activation. Furthermore, inhibition of the purinergic receptor P2Y12 attenuated HMGB1-dependent platelet activation. Mechanistically, we show that HMGB1 stimulated ADP secretion, while concomitantly increasing P2Y12 levels at the platelet membrane. We show that in SCD patients, increased plasma HMGB1 levels were associated with heightened platelet activation and surface P2Y12 expression. Treatment of healthy platelets with plasma from SCD patients enhanced platelet activation and surface P2Y12, and increased sensitivity to ADP-mediated activation, and these effects were linked to plasma HMGB1. We conclude that HMGB1-mediated platelet activation involves ADP-dependent P2Y12 signaling, and HMGB1 primes platelets for ADP signaling. This complementary agonism between ADP and HMGB1 furthers the understanding of thromboinflammatory signaling in conditions such as SCD, and provides insight for therapeutic P2Y12 inhibition.

TEMPORARY REMOVAL: Alterations in visible light exposure modulate platelet function and regulate thrombus formation.

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High Dose of Metformin Decreases Susceptibility to Occlusive Arterial Thrombosis in Diabetic Mice.

Introduction: Metformin is the most prescribed medication in Diabetes Mellitus(DM). Metformin has shown to decrease mean platelet volume, with promising antiplatelet effects. High doses of Metformin have also been associated with hypercoagulation. We hypothesize that Metformin will protect DM mice from occlusive arterial thrombus formation by altering platelet activation and mitochondrial bioenergetics. Methods: DM was developed by low dose of Streptozotocin, healthy (non-DM) mice are controls. Either vehicle or Metformin was administered twice daily via oral gavage for 7-days. Ferric chloride (FeCl3) arterial thrombosis and tail bleeding time were performed. Whole blood aggregometry, platelet activation/adhesion and mitochondrial bioenergetics were evaluated. Results: Metformin decreased susceptibility of DM mice to arterial thrombosis. Platelet bioenergetics show DM mice have increased platelet mitochondrial respiration, but no differences were observed with Metformin treatment. In healthy mice, Metformin modulated ADP-dependent increase in platelet adhesion. In healthy mice, Metformin shortens bleeding time with faster thrombotic occlusion. Metformin also increased platelet mitochondrial maximal respiration and spare respiratory capacity uniquely in healthy mice. Conclusion: Metformin regulates platelet bioenergetics and ADP-mediated platelet function in DM mice which attenuates susceptibility to arterial thrombosis. Future studies will evaluate clinically relevant doses of Metformin that regulates thrombotic function in diabetic platelets.

High Dose of Metformin Decreases Susceptibility to Occlusive Arterial Thrombosis in Diabetic Mice.

Introduction: Metformin is the most prescribed medication in Diabetes Mellitus(DM). Metformin has shown to decrease mean platelet volume, with promising antiplatelet effects. High doses of Metformin have also been associated with hypercoagulation. We hypothesize that Metformin will protect DM mice from occlusive arterial thrombus formation by altering platelet activation and mitochondrial bioenergetics. Methods: DM was developed by low dose of Streptozotocin, non-DM (healthy) mice are controls. Either vehicle or Metformin was administered twice daily via oral gavage for 7-days. Ferric chloride (FeCl3) arterial thrombosis and tail bleeding time were performed. Whole blood aggregometry, platelet activation/adhesion and mitochondrial bioenergetics were evaluated. Results: Metformin decreased susceptibility of DM mice to arterial thrombosis. Platelet bioenergetics show DM mice have increased platelet mitochondrial respiration, but no differences were observed with Metformin treatment. In non-DM (healthy) mice, Metformin modulated ADP-dependent increase in platelet adhesion. Non-DM (healthy) mice, Metformin shortens bleeding time with faster thrombotic occlusion. Metformin also increased platelet mitochondrial maximal respiration and spare respiratory capacity uniquely in non-DM (healthy) mice. Conclusion: Metformin regulates platelet bioenergetics and ADP-mediated platelet function in DM mice which attenuates susceptibility to arterial thrombosis. Future studies will evaluate clinically relevant doses of Metformin that regulates thrombotic function in diabetic platelets.

Release of hepatic xanthine oxidase (XO) to the circulation is protective in intravascular hemolytic crisis.

Xanthine oxidase (XO) catalyzes the catabolism of hypoxanthine to xanthine and xanthine to uric acid, generating oxidants as a byproduct. Importantly, XO activity is elevated in numerous hemolytic conditions including sickle cell disease (SCD); however, the role of XO in this context has not been elucidated. Whereas long-standing dogma suggests elevated levels of XO in the vascular compartment contribute to vascular pathology via increased oxidant production, herein, we demonstrate, for the first time, that XO has an unexpected protective role during hemolysis. Using an established hemolysis model, we found that intravascular hemin challenge (40 µmol/kg) resulted in a significant increase in hemolysis and an immense (20-fold) elevation in plasma XO activity in Townes sickle cell phenotype (SS) sickle mice compared to controls. Repeating the hemin challenge model in hepatocyte-specific XO knockout mice transplanted with SS bone marrow confirmed the liver as the source of enhanced circulating XO as these mice demonstrated 100% lethality compared to 40% survival in controls. In addition, studies in murine hepatocytes (AML12) revealed hemin mediates upregulation and release of XO to the medium in a toll like receptor 4 (TLR4)-dependent manner. Furthermore, we demonstrate that XO degrades oxyhemoglobin and releases free hemin and iron in a hydrogen peroxide-dependent manner. Additional biochemical studies revealed purified XO binds free hemin to diminish the potential for deleterious hemin-related redox reactions as well as prevents platelet aggregation. In the aggregate, data herein reveals that intravascular hemin challenge induces XO release by hepatocytes through hemin-TLR4 signaling, resulting in an immense elevation of circulating XO. This increased XO activity in the vascular compartment mediates protection from intravascular hemin crisis by binding and potentially degrading hemin at the apical surface of the endothelium where XO is known to be bound and sequestered by endothelial glycosaminoglycans (GAGs).

Fibrinogen Mitigates Prion-Mediated Platelet Activation and Neuronal Cell Toxicity.

Prion peptide (PrP) misfolds to infectious scrapie isoform, the ß pleat-rich insoluble fibrils responsible for neurodegeneration and fatal conformational diseases in humans. The amino acid sequence 106-126 from prion proteins, PrP(106-126), is highly amyloidogenic and implicated in prion-induced pathologies. Here, we report a novel interaction between PrP(106-126) and the thrombogenic plasma protein fibrinogen that can lead to mitigation of prion-mediated pro-thrombotic responses in human platelets as well as significant decline in neuronal toxicity. Thus, prior exposure to fibrinogen-restrained PrP-induced rise in cytosolic calcium, calpain activation, and shedding of extracellular vesicles in platelets while it, too, averted cytotoxicity of neuronal cells triggered by prion peptide. Interestingly, PrP was found to accelerate fibrin-rich clot formation, which was resistant to plasmin-mediated fibrinolysis, consistent with enhanced thrombus stability provoked by PrP. We propose that PrP-fibrinogen interaction can be clinically exploited further for prevention and management of infectious prion related disorders. Small molecules or peptides mimicking PrP-binding sites on fibrinogen can potentially mitigate PrP-induced cellular toxicity while also preventing the negative impact of PrP on fibrin clot formation and lysis.

Mitochondrial reactive oxygen species scavenging attenuates thrombus formation in a murine model of sickle cell disease.

BACKGROUND: Sickle cell disease (SCD) is characterized by hemolysis-associated platelet dysfunction that leads to increased risk of thrombosis and plays a role in the high morbidity and mortality of the disease. The mechanisms by which hemolysis induces platelet activation remain unclear. We recently demonstrated that patients with SCD showed increased platelet mitochondrial reactive oxygen species (mtROS) production that correlates with markers of hemolysis and platelet activation. Experiments in isolated platelets demonstrated that mtROS stimulated platelet activation. However, the role of hemolysis-induced mtROS in thrombus formation in vivo remains unclear. OBJECTIVES: Here, we hypothesize that scavenging of mtROS attenuates the propensity for thrombosis in mouse models of hemolysis. METHODS: We used models of hemolysate infusion into wildtype mice as well as the Berkley transgenic mouse model of SCD, a chronic mode of hemolysis, to test the effect of hemolysis on platelet mtROS production and thrombosis. RESULTS: We show that infusion of hemolysate in wildtype mice induces platelet mtROS production and decreases time to vessel occlusion in a model of ferric chloride-induced carotid artery thrombosis. Increased mtROS and propensity for thrombosis was also observed in the Berkley transgenic mouse model of SCD. Notably, treatment with mtROS scavengers decreased platelet mtROS levels and attenuated the propensity for thrombus formation in both models. CONCLUSIONS: These data demonstrate that mtROS significantly contribute to the mechanism of hemolysis-induced thrombosis in vivo and suggest a potential role for mitochondrially targeted antioxidant therapy in hemolysis and SCD-related thrombosis.