Merrill-Skoloff, G. | Search bioRxiv

bioRxiv2025

POmAb, an antibody targeting open prothrombin, results in anticoagulation without excessive bleeding in mice

Anti-prothrombin antibodies are commonly found in patients with Antiphospholipid Syndrome (APS), yet their role in clinical manifestations remains unclear. We recently identified two classes of anti-prothrombin antibodies based on their ability to recognize closed and open forms of prothrombin. Type-I antibodies bind to the open form, while Type-II antibodies bind to both forms. POmAb is a prototypical Type-I antibody that specifically targets kringle-1 of prothrombin, maintaining it in an open state. In this study, we assess the effects of POmAb in mice using the cremaster arteriole laser-induced injury model. POmAb bound mouse prothrombin and decreased thrombin generation in mouse plasma. When administered intravenously shortly before the injury, POmAb quickly accumulated on the damaged vessel wall. This accumulation significantly reduced fibrin generation with a modest effect on platelet accumulation and without causing excessive bleeding. Results obtained with POmAb offer insights into the potential roles of the anti-prothrombin antibodies in APS. They also provide proof of concept for a new class of anticoagulants that, by specifically targeting open prothrombin, could mitigate thrombosis with reduced bleeding risk.

molecular biology↗

bioRxiv2021

The secreted tyrosine kinase VLK is essential for normal platelet activation and thrombus formation

Tyrosine phosphorylation of extracellular proteins is observed in cell cultures and in vivo, but little is known about the functional roles of tyrosine phosphorylation of extracellular proteins. Vertebrate Lonesome Kinase (VLK) is a broadly expressed secretory pathway tyrosine kinase present in platelet [a]-granules. It is released from platelets upon activation and phosphorylates substrates extracellularly. Its role in platelet function, however, has not been previously studied. In human platelets, we identified phosphorylated tyrosines mapped to luminal or extracellular domains of transmembrane and secreted proteins implicated in the regulation of platelet activation. To determine the role of VLK in extracellular tyrosine phosphorylation and platelet function, we generated mice with a megakaryocyte/platelet-specific deficiency of VLK. Platelets from these mice are normal in abundance and morphology, but have dramatic changes in function both in vitro and in vivo. Resting and thrombin-stimulated VLK-deficient platelets demonstrate a significant decrease of several tyrosine phosphobands. Functional testing of VLK-deficient platelets shows decreased PAR4- and collagen-mediated platelet aggregation, but normal responses to ADP. Dense granule and -granule release are reduced in these platelets. Furthermore, VLK-deficient platelets exhibit decreased PAR4-mediated Akt (S473) and Erk1/2(T202/Y204) phosphorylation, indicating altered proximal signaling. In vivo, mice lacking VLK in megakaryocytes/platelets demonstrate strongly reduced platelet accumulation and fibrin formation following laser-injury of cremaster arterioles compared to controls. These studies demonstrate that the secretory pathway tyrosine kinase VLK is critical for stimulus-dependent platelet activation and thrombus formation, providing the first evidence that a secreted protein kinase is required for normal platelet function.

cell biology↗