OC 43.4 - Plasmin-cleaved VWF: a biomarker for microthrombosis

Background: Von Willebrand factor (VWF) is an essential contributor to hemostasis, but also to arterial and microvascular thrombosis. VWF is cleaved by ADAMTS13 to limit its prothrombotic properties. Failure to do so can lead to microthrombosis as is seen in thrombotic thrombocytopenic purpura (TTP). In this setting, plasmin becomes active to cleave VWF, as we previously demonstrated during acute TTP attacks in human patients. Furthermore, induction of plasmin activity attenuates TTP pathogenesis in preclinical models in vivo. These combined findings suggest that plasmin is an endogenous regulator of VWF thrombogenicity. We therefore hypothesize that assessment of plasmin-cleaved VWF (cVWF) reflects microvascular thrombosis (analogous to D-dimer for VTE). However, no assays are available to investigate cVWF in pathophysiology.

Aims: To develop a bioassay to track plasmin-mediated VWF cleavage in plasma.

Methods: Plasma-purified VWF was cleaved with plasmin and characterized by Western blotting. Nanobodies (llama-derived antibody fragments) were developed to discriminate between cVWF and intact VWF by phage display.

Results: We developed an ELISA-based bioassay that recognizes cVWF, generated after plasmin cleavage. The product that is captured was characterized by Western blotting and consists of three characteristic disulfide-linked main cleavage fragments (140, 110 and 70 kDa; under reducing conditions). The same products are also formed upon plasmin-mediated destruction of platelet-VWF microthrombi in vitro. In spiked citrated plasma, cVWF was detected with high sensitivity at concentrations (lower detection limit 100 ng/mL) that cannot be detected by Western blotting. Validations in TTP patient samples are currently ongoing.

Conclusion(s): Our studies demonstrate that cVWF can be discriminated from intact VWF in an ELISA-based setup. Detection of cVWF may serve as a biomarker for pathologies involving microvascular thrombosis such as TTP, and possibly other diseases.