The platelet receptors glycoprotein Ibα (GPIbα) and GPVI are known to be cleaved by members of a disintegrin and metalloprotease (ADAM) family (ADAM10 and ADAM17), but the mechanisms and consequences of this shedding are not well understood. Our results revealed that (1) glycoprotein shedding is confined to distinct platelet populations showing near-complete shedding, (2) the heterogeneity between (non)shed platelets is independent of agonist type but coincides with exposure of phosphatidylserine (PS), and (3) distinct pathways of shedding are induced by elevated Ca, low Ca protein kinase C (PKC), or apoptotic activation. Furthermore, we found that receptor shedding reduces binding of von Willebrand factor, enhances binding of coagulation factors, and augments fibrin formation. In response to Ca-increasing agents, shedding of GPIbα was abolished by ADAM10/17 inhibition but not by blockage of calpain. Stimulation of PKC induced shedding of only GPIbα, which was annulled by kinase inhibition. The proapoptotic agent ABT-737 induced shedding, which was caspase dependent. In Scott syndrome platelets that are deficient in Ca-dependent PS exposure, shedding occurred normally, indicating that PS exposure is not a prerequisite for ADAM activity. In whole-blood thrombus formation, ADAM-dependent glycoprotein shedding enhanced thrombin generation and fibrin formation. Together, these findings indicate that 2 major activation pathways can evoke ADAM-mediated glycoprotein shedding in distinct platelet populations and that shedding modulates platelet function from less adhesive to more procoagulant.

Patients suffering from chronic kidney disease (CKD) are at a 20-fold higher risk of dying due to cardiovascular diseases (CVDs), primarily thrombosis following vascular injury. CKD is connected with retention of uremic toxins, especially indoxyl sulfate (IS), which are currently considered as a non-classical CKD-specific risk factor for CVDs. The present study aimed to examine the effect of chronic exposure to IS on the hemostatic system and arterial thrombosis in a model without greater interferences from the uremic milieu consisting of additional uremic toxins. Forty-eight male Wistar Crl:WI (cmdb) rats were divided into three groups: one control group and two experimental groups, which were exposed to 100 or 200 mg/kg of b.w./day of IS in drinking water for a period of 28 days. The control group received water without IS. At the end of the experiment, the induction of arterial thrombosis was performed. We investigated the impact of IS on thrombosis incidence, kinetics and strength of clot formation, platelet activity, aortic contents of sirtuin (SIRT) 1 and sirtuin 3 (SIRT3), hemostatic system, cardiorespiratory parameters, biochemistry of plasma and urine as well as histology of the thrombus, kidney, and liver. Obtained data revealed that chronic exposure to IS promotes arterial thrombosis via increased levels of complex tissue factor/factor VII, plasminogen activator inhibitor-1 (PAI-1), platelet activation, as well as decreased aortic levels of SIRT1 and SIRT3. Therefore, we hypothesize that IS enhances primary hemostasis leading to augmented formation of platelet plug with increased amounts of fibrin and affects secondary hemostasis through the influence on plasma coagulation and fibrinolysis factors, which results in the increased kinetics and strength of clot formation. The findings described may contribute to a better understanding of the mechanisms leading to increased thrombotic events in patients with CKD with elevated levels of IS.

Chronic kidney disease (CKD) patients are at high risk for thrombotic events. Indoxyl sulfate (IS) is one of the most potent uremic toxins that accumulates during CKD. Even though IS is associated with an increased risk for cardiovascular disease, its impact on thrombotic events still remains not fully understood. The purpose of the study was to evaluate the direct effect of IS on thrombotic process. We examined the impact of acute exposure to IS on thrombus development induced by electric current in Wistar rats, intravital thrombus formation after laser-induced injury in the mice endothelium, coagulation profile, clot formation dynamics, platelet aggregations, and erythrocyte osmotic resistance. IS doses: 10, 30 and 100 mg/kg body weight (b.w.) increased weight of thrombus induced by electric current in dose-dependent manner (p < 0.001). Furthermore, two highest IS doses increased laser-induced thrombus formation observed via confocal system (increase in fluorescence intensity and total thrombus area (p < 0.01)). Only the highest IS dose decreased clotting time (p < 0.01) and increased maximum clot firmness (p < 0.05). IS did not affect blood morphology parameters and erythrocyte osmotic resistance, but augmented collagen-induced aggregation. Obtained data indicate that IS creates prothrombotic state and contributes to more stable thrombus formation. Thus, we concluded that IS may be one of crucial uremic factors promoting thrombotic events in CKD patients.

Ultra-low-field (ULF) nuclear magnetic resonance (NMR) is a promising spectroscopy method allowing for, e.g., the simultaneous detection of multiple nuclei. To overcome the low signal-to-noise ratio that usually hampers a wider application, we present here an alternative approach to ULF NMR, which makes use of the hyperpolarizing technique signal amplification by reversible exchange (SABRE). In contrast to standard parahydrogen hyperpolarization, SABRE can continuously hyperpolarize 1 H as well as other MR-active nuclei. For simultaneous measurements of 1 H and 19 F under SABRE conditions a superconducting quantum interference device (SQUID)-based NMR detection unit was adapted. We successfully hyperpolarized fluorinated pyridine derivatives with an up to 2000-fold signal enhancement in 19 F. The detected signals may be explained by two alternative reaction mechanisms. SABRE combined with simultaneous SQUID-based broadband multinuclear detection may enable the quantitative analysis of multinuclear processes.