Heurich, M. | bioRxiv Science

bioRxiv2021

Complement Regulator Factor H is a Cofactor for Thrombin in both Pro- and Anticoagulant Roles

BackgroundComplement FH (FH) is a key regulator of complement activity whereas thrombin (FIIa) is central to hemostasis with both pro- and anticoagulant functions. Both have separately been shown to have auxiliary activities across the two systems. The purpose of this study was to determine the effect of FH on pro- and anti-coagulant functions and investigate the interaction between FH and thrombin. MethodsTail bleeding time and hemolysis were measured in FH-deficient mice (CFH-/-). Activated partial thromboplastin time (aPTT) was determined in FH-depleted human plasma. FH effect on fibrin clot generation was investigated in turbidity assays and on activated protein C (APC) generation. Binding affinity of thrombin with FH was determined using surface plasmon resonance (SPR). ResultsTail bleeding time in CFH-/- mice was significantly prolonged compared to wild type mice. The aPTT in FH-depleted human plasma was elevated compared to normal plasma and restored by adding back FH to depleted plasma. Accordingly, FH enhanced thrombin-mediated fibrin clot generation by shortening lag time, increasing rate of clot formation and maximum turbidity, and affected clot structure. Despite this, FH also increased the rate of thrombin-mediated protein C (PC) activation, both in the presence and absence of soluble recombinant thrombomodulin (TM). Nanomolar affinity binding of FH with thrombin, but not prothrombin, was confirmed. ConclusionComplement FH binds thrombin with strong affinity and acts as a novel cofactor that enhances both pro- and anticoagulant actions of thrombin. These data highlight an important role for FH in hemostasis. Key pointsO_LIAbsence of FH prolongs tail bleeding time in CFH-/- mice and activated partial thromboplastin time (aPTT) is elevated in human FH-depleted plasma. C_LIO_LIFH acts a cofactor for thrombin by enhancing fibrin generation, altering fibrin clot structure and enhancing TM-thrombin mediated protein C activation C_LI

immunology↗

bioRxiv2021

The Structure of ChAdOx1/AZD-1222 Reveals Interactions with CAR and PF4 with Implications for Vaccine-induced Immune Thrombotic Thrombocytopenia

Adenovirus derived vectors, based on chimpanzee adenovirus Y25 (ChAdOx1) and human adenovirus type 26 are proving critical in combatting the 2019 SARS-CoV-2 pandemic. Following emergency use authorisation, scale up in vaccine administration has inevitably revealed vaccine related adverse effects; too rare to observe even in large Phase-III clinical trials. These include vaccine-induced thrombotic thrombocytopenia (VITT), an ultra-rare adverse event in which patients develop life-threatening blood clots 5-24 days following vaccination. To investigate vector-host interactions of ChAdOx1 underpinning VITT we solved the structure of the ChAdOx1 capsid by CryoEM, and the structure of the primary receptor tropism determining fiber-knob protein by crystallography. These structural insights have enabled us to unravel key protein interactions involved in ChAdOx1 cell entry and a possible means by which it may generate misplaced immunity to platelet factor 4 (PF4), a protein involved in coagulation. We use in vitro cell binding assays to show that the fiber-knob protein uses coxsackie and adenovirus receptor (CAR) as a high affinity binding partner, while it does not form a stable interface with CD46. Computational simulations identified a putative mechanism by which the ChAdOx1 capsid interacts with PF4 by binding in the spaces between hexon proteins, with downstream implications for the causes of VITT. SummaryWe present the structure of the ChAdOx1 viral vector, derived from chimpanzee adenovirus Y25 at 4.2[A] resolution1. ChAdOx1 is in global use in the AstraZeneca vaccine, ChAdOx1 nCoV-19/AZD-1222, to combat the SARS-CoV-2 coronavirus pandemic. Recently observed, rare, adverse events make detailed mechanistic understanding of this vector key to informing proper treatment of affected patients and the development of safer viral vectors. Here, we determine a primary mechanism ChAdOx1 uses to attach to cells is coxsackie and adenovirus receptor (CAR), a protein which is identical in humans and chimpanzees. We demonstrate the vector does not form a stable CD46 interaction, a common species B adenovirus receptor, via its primary attachment protein. Further, we reveal the surface of the ChAdOx1 viral capsid has a strong electronegative potential. Molecular simulations suggest this charge, together with shape complementarity, are a mechanism by which an oppositely charged protein, platelet factor 4 (PF4) may bind the vector surface. PF4 is a key protein involved in the formation of blood clots2, and the target of auto-antibodies in heparin-induced immune thrombotic thrombocytopenia (HITT)3, an adverse reaction to heparin therapy which presents similarly to vaccine-induced immune thrombotic thrombocytopenia (VITT), a rare complication of ChAdOx1 nCoV-19 vaccination4-6. We propose a mechanism in which the ChAdOx1-PF4 complex may stimulate the production of antibodies against PF4, leading to delayed blood clot formation, as observed in VITT.

molecular biology↗