Functional Analysis Of TTP-Associated ADAMTS13 Mutants Under Shear Flow

Upon agonist stimulation endothelial cells secrete ultralarge von Willebrand Factor (VWF) multimers. These multimers form prothrombotic VWF strings on the cell's surface which are size-regulated by ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13). Under physiological conditions, this metalloprotease cleaves VWF between Tyr¹⁶⁰⁵ and Met¹⁶⁰⁶ in its A2 domain only when this domain is unfolded by shear stress and the respective binding sites of both, VWF and ADAMTS13, adjoin each other (Gao W, et al., Blood. 2008 Sep 1;112(5):1713-9). This sophisticated substrate recognition involves distinct steps during which the TSP5-CUB domains of ADAMTS13 initially bind to VWF shear force independent, then, upon shear induced elongation of VWF, additional exosite binding sites become exposed which subsequently can be bound by the complementary spacer domain of ADAMTS13 (Crawley JT, et al., Blood. 2011 Sep 22;118(12):3212-21).

Mutations in ADAMTS13 cause Upshaw-Schulman Syndrome, the inherited form of Thrombotic Thrombocytopenic Purpura (TTP), a microangiopathy characterized by platelet clumping, hemolytic anemia, and subsequent organ failure (Coppo P & Veyradier A, Cardiovasc Hematol Disord Drug Targets. 2009 Mar;9(1):36-50).

State-of-the-art ADAMTS13 tests of patients' plasma use static approaches based on either proteolysis of VWF minimal fragments or full length VWF under denaturing conditions. In some cases the results of these static assays could be without pathological finding, e.g. when a mutation in the shear dependent spacer region of ADAMTS13 would prevent binding to VWF strings only under flow conditions without affecting its proteolytic activity towards denatured VWF substrates in static assays.

We developed an assay based on gain of function GPIba mutants immobilized on latex particles that facilitates the visualization of VWF strings on the surface of stimulated endothelial cells without the need for whole blood or isolated platelets. We then used this assay to observe the proteolytic activity of wildtype (wt) ADAMTS13. Under shear flow imitating the physiological conditions of the venous blood stream, VWF string formation was induced by histamine stimulation and visualized by binding of the GPIba coated particles. wtADAMTS13 was then floated over the cells at the same shear rate (500 s^-1) thereby offering the VWF strings as substrate. VWF string proteolysis was observed live and the time was determined that was needed for complete degradation. wtADAMTS13 degraded the up to 1mm long VWF strings within seconds. 5-10 minutes after ADAMTS13 addition only a few string fragments with a maximal length of 10 µm remained intact. To determine the accuracy of our assay, we then determined the catalytic activity of two ADAMTS13 mutants with mutations in the C-terminal TPS1 domains. Since these domains are responsible for shear flow independent substrate binding we expected the same results as in the static assays. Mutant ADAMTS13-R1060W possesses a single point-mutation in its TSP1-7 domain which leads to a secretion defect and a catalytic activity that is reduced by 65% in a static assay. We indeed also determined a decrease in proteolytic activity by 65% and VWF multimeric fragments with a size of 10-30 µm remained intact. The truncated mutant ADAMTS13-R910*, lacking the TSP1-7, TSP1-8 and the CUP domains was incapable of producing VWF-fragments smaller than 80 µm within one hour and therefore only showed a minimal residual activity in accordance to the previously published catalytic activity of 2%.

After these prove-of-principle experiments our assay could now be adapted to be used with blood samples of TTP patients who exhibit reduced proteolytic band patterns in VWF multimer analysis but possess normal ADAMTS13 activity in static assays.

Summarizing we developed an assay that allows functional characterization of TTP-associated ADAMTS13 mutants with shear flow dependent dis-functionalities. The assay may serve as a valuable tool for research but could also broaden the range of TTP diagnostics.