Deciphering and disrupting PIEZO1-TMEM16F interplay in hereditary xerocytosis.

ABSTRACT: Cell-surface exposure of phosphatidylserine (PS) is essential for phagocytic clearance and blood clotting. Although a calcium-activated phospholipid scramblase (CaPLSase) has long been proposed to mediate PS exposure in red blood cells (RBCs), its identity, activation mechanism, and role in RBC biology and disease remain elusive. Here, we demonstrate that TMEM16F, the long-sought-after RBC CaPLSase, is activated by calcium influx through the mechanosensitive channel PIEZO1 in RBCs. PIEZO1-TMEM16F functional coupling is enhanced in RBCs from individuals with hereditary xerocytosis (HX), an RBC disorder caused by PIEZO1 gain-of-function channelopathy. Enhanced PIEZO1-TMEM16F coupling leads to an increased propensity to expose PS, which may serve as a key risk factor for HX clinical manifestations including anemia, splenomegaly, and postsplenectomy thrombosis. Spider toxin GsMTx-4 and antigout medication benzbromarone inhibit PIEZO1, preventing force-induced echinocytosis, hemolysis, and PS exposure in HX RBCs. Our study thus reveals an activation mechanism of TMEM16F CaPLSase and its pathophysiological function in HX, providing insights into potential treatment.

Clot Imaging Using Photostable Nanodiamond.

While thrombosis is the leading cause of morbidity and mortality in the United States, an understanding of its triggers, progression, and response to anticoagulant therapy is lacking. Intravital fluorescence microscopy has advanced the study of thrombus formation by providing targeted, multi-color contrast. However, photodegradation of fluorophores limits the application in longitudinal studies (e.g., clot progression and/or dissolution). Fluorescent nanodiamond (FND) is a fluorophore which utilizes intrinsic fluorescence of chromogenic centers within and protected by the diamond crystalline lattice. Recent developments in diamond processing have allowed for the controlled production of nanodiamonds emitting in green or red. Here, the use of FND to label blood clots and/or clot lysis is demonstrated and compared to commonly used organic fluorophores. Model ex vivo clots were formed with incorporated labeled fibrinogen to allow imaging. FND was shown to match the morphology of organic fluorophore labels absent of photobleaching over time. The addition of tissue plasminogen activator (tPa) allowed visualization of the clot lysis stage, which is vital to studies of both DVT and pulmonary embolism resolution.

Minimal role for the alternative pathway in complement activation by HIT immune complexes.

BACKGROUND: Anti-platelet factor 4 (PF4)/heparin immune complexes that cause heparin-induced thrombocytopenia (HIT) activate complement via the classical pathway. Previous studies have shown that the alternative pathway of complement substantially amplifies the classical pathway of complement activation through the C3b feedback cycle. OBJECTIVES: These studies sought to examine the contributions of the alternative pathway to complement activation by HIT antibodies. METHODS: Using IgG monoclonal (KKO) and/or patient-derived HIT antibodies, we compared the effects of classical pathway (BBK32 and C1-esterase inhibitor [C1-INH]), alternative pathway (anti-factor B [fB] or factor D [fD] inhibitor) or combined classical and alternative pathway inhibition (soluble complement receptor 1 [sCR1]) in whole blood or plasma. RESULTS: Classical pathway inhibitors BBK32 and C1-INH and the combined classical/alternative pathway inhibitor sCR1 prevented KKO/HIT immune complex-induced complement activation, including release of C3 and C5 activation products, binding of immune complexes to B cells, and neutrophil activation. The alternative pathway inhibitors fB and fD, however, did not affect complement activation by KKO/HIT immune complexes. Similarly, alternative pathway inhibition had no effect on complement activation by unrelated immune complexes consisting of anti-dinitrophenyl (DNP) antibody and the multivalent DNP--keyhole limpet hemocyanin antigen. CONCLUSIONS: Collectively, these findings suggest the alternative pathway contributes little in support of complement activation by HIT immune complexes. Additional in vitro and in vivo studies are required to examine if this property is shared by most IgG-containing immune complexes or if predominance of the classic pathway is limited to immune complexes composed of multivalent antigens.