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Background:  Protein Z-dependent protease inhibitor (ZPI) is an anticoagulant serpin that targets factor Xa (FXa) in the presence of protein Z (PZ), and factor XIa (FXIa). In factor-VIII-deficient mice, PZ or ZPI gene knock-out mitigates the bleeding phenotype, and pharmacological inhibition of PZ enhances thrombin generation in plasma from patients with hemophilia., Aims:  To develop a single-domain antibody (sdAb) directed against ZPI to inhibit its anticoagulant activity., Methods:  We screened for anti-ZPI sdAbs in a llama-derived phage display immune library of sdAbs. The sdAbs that bound ZPI were produced and purified for characterization. The binding of sdAbs to ZPI or other serpins was evaluated using ELISAs, and ZPI inhibition was measured in an anti-FXa or anti-FXIa chromogenic assay. The sdAbs's procoagulant activity was assessed in a thrombin generation assay in normal plasma, factor VIII- and FXI-deficient plasma., Results:  Of the four sdAbs found to bind to ZPI, one (referred to as ZPI-sdAb2) dose-dependently inhibited ZPI's anti-FXa and anti-FXIa activities with a mean half-maximal inhibitory concentration of 1.8 and 1.3 µM, respectively. ZPI-sdAb2 did not cross-react with other plasma serpins, such as antithrombin and α1-antitrypsin. ZPI-sdAb2 induced a significant increase in thrombin generation in plasma samples from healthy donors, patients with severe hemophilia A, and patients with FXI deficiency., Conclusion:  ZPI-sdAb2 is the first specific, direct ZPI inhibitor found to exhibit procoagulant activity in plasma. This sdAb might have potential as a treatment for hemophilia or other bleeding disorders., Competing Interests: None declared., (Thieme. All rights reserved.)

Septic shock is the archetypal clinical setting in which extensive crosstalk between inflammation and coagulation dysregulates the latter. The main anticoagulant systems are systematically impaired, depleted, and/or downregulated. Protein Z-dependent protease inhibitor (ZPI) is an anticoagulant serpin that not only targets coagulation factors Xa and XIa but also acts as an acute phase reactant whose plasma concentration rises in inflammatory settings. The objective of the present study was to assess the plasma ZPI antigen level in a cohort of patients suffering from septic shock with or without overt-disseminated intravascular coagulation (DIC). The plasma ZPI antigen level was approximately 2.5-fold higher in the patient group ( n  = 100; 38 with DIC and 62 without) than in healthy controls ( n  = 31). The elevation's magnitude did not appear to depend on the presence/absence of DIC. Furthermore, Western blots revealed the presence of cleaved ZPI in plasma from patients with severe sepsis, independently of the DIC status. In vitro, ZPI was proteolytically inactivated by purified neutrophil elastase (NE) and by NE on the surface of neutrophil extracellular traps (NETs). The electrophoretic pattern of ZPI after NE-catalyzed proteolysis was very similar to that resulting from the clotting process-suggesting that the cleaved ZPI observed in severe sepsis plasma is devoid of anticoagulant activity. Taken as a whole, our results (1) suggest that NE is involved in ZPI inactivation during sepsis, and (2) reveal a novel putative mechanism for the procoagulant activity of NETs in immunothrombosis., Competing Interests: None declared., (Thieme. All rights reserved.)

The multimeric plasma glycoprotein (GP) von Willebrand factor (VWF) is best known for recruiting platelets to sites of injury during primary hemostasis. Generally, mutations in the VWF gene lead to loss of hemostatic activity and thus the bleeding disorder von Willebrand disease. By employing cone and platelet aggregometry and microfluidic assays, we uncovered a platelet GPIIb/IIIa-dependent prothrombotic gain of function (GOF) for variant p.Pro2555Arg, located in the C4 domain, leading to an increase in platelet aggregate size. We performed complementary biophysical and structural investigations using circular dichroism spectra, small-angle X-ray scattering, nuclear magnetic resonance spectroscopy, molecular dynamics simulations on the single C4 domain, and dimeric wild-type and p.Pro2555Arg constructs. C4-p.Pro2555Arg retained the overall structural conformation with minor populations of alternative conformations exhibiting increased hinge flexibility and slow conformational exchange. The dimeric protein becomes disordered and more flexible. Our data suggest that the GOF does not affect the binding affinity of the C4 domain for GPIIb/IIIa. Instead, the increased VWF dimer flexibility enhances temporal accessibility of platelet-binding sites. Using an interdisciplinary approach, we revealed that p.Pro2555Arg is the first VWF variant, which increases platelet aggregate size and shows a shear-dependent function of the VWF stem region, which can become hyperactive through mutations. Prothrombotic GOF variants of VWF are a novel concept of a VWF-associated pathomechanism of thromboembolic events, which is of general interest to vascular health but not yet considered in diagnostics. Thus, awareness should be raised for the risk they pose. Furthermore, our data implicate the C4 domain as a novel antithrombotic drug target., Competing Interests: None declared., (The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution-NonDerivative-NonCommercial License, permitting copying and reproduction so long as the original work is given appropriate credit. Contents may not be used for commercial purposes, or adapted, remixed, transformed or built upon. (https://creativecommons.org/licenses/by-nc-nd/4.0/).)