Plasmin-cleaved von Willebrand factor as a biomarker for microvascular thrombosis.

ABSTRACT: von Willebrand factor (VWF) is an essential contributor to microvascular thrombosis. Physiological cleavage by ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) limits its prothrombotic properties, explaining why ADAMTS13 deficiency leads to attacks of microthrombosis in patients with thrombotic thrombocytopenic purpura (TTP). We previously reported that plasminogen activation takes place during TTP attacks in these patients. Furthermore, stimulation of plasminogen activation attenuates pathogenesis in preclinical TTP models in vivo. This suggests that plasmin is an endogenous regulator of VWF thrombogenicity, in particular when ADAMTS13 falls short to prevent microvascular occlusions. VWF cleavage by plasmin is biochemically distinct from cleavage by ADAMTS13. We hypothesized that plasmin-cleaved VWF (cVWF) holds value as a biomarker of microvascular thrombosis. Here, we describe the development of a variable domain of heavy-chain-only antibody (VHH)-based bioassay that can distinguish cVWF from intact and ADAMTS13-cleaved VWF in plasma. We validate this assay by tracking cVWF release during degradation of microthombi in vitro. We demonstrate that endogenous cVWF formation takes place in patients with TTP during acute attacks of thrombotic microangiopathy but not in those in remission. Finally, we show that therapeutic plasminogen activation in a mouse model of TTP amplifies cVWF formation, which is accompanied by VWF clearance. Our combined findings indicate that cVWF is released from microthrombi in the context of microvascular occlusion.

FXII contact activation is dependent on binding through two unique patches in its EGF-1 domain and can be prevented by (VHH) targeting.

BACKGROUND: Factor XII (FXII) triggers contact activation by binding to foreign surfaces, with the EGF-1 domain being the primary binding site. Blocking FXII surface-binding might hold therapeutic value to prevent medical device-induced thrombosis. OBJECTIVES: To unravel and prevent EGF-1 mediated FXII surface-binding with VHH. METHODS: FXII variants with glutamine substitutions of two positively-charged amino acid patches within the EGF-1 domain were created. Their role in FXII contact activation was assessed using kaolin pull-down experiments, amidolytic activity assays, and clotting assays. FXII EGF-1 domain specific VHHs were raised to inhibit EGF-1 mediated FXII contact activation while preserving quiescence. RESULTS: Two unique, positively-charged patches in the EGF1 domain were identified (upstream: 73K74K76K78H81K82H; downstream: 87K113K). Neutralizing the charge of both patches led to a 99% reduction in FXII kaolin binding, subsequent decrease in auto-activation of 94% and prolongation of clot formation in aPTT assays from 36 (±2) to 223 (±13) seconds. Three FXII EGF-1 specific VHHs were developed, that are capable of inhibiting kaolin binding and subsequent contact system activation in plasma. The most effective V HH 'F2' binds the positively-charged patches and thereby dose-dependently extends aPTT clotting times from 29 (±2) to 43 (±3) seconds without disrupting FXII quiescence. CONCLUSION: The two unique, positively-charged patches in FXII EGF-1 cooperatively mediate FXII surface-binding making both patches crucial for contact activation. Targeting these with FXII EGF-1 specific VHHs can exclusively decrease FXII surface-binding and subsequent contact activation, while preserving zymogen quiescence. These patches thus have potential as druggable target in preventing medical device-induced thrombosis.

Factor XII Explored with AlphaFold - Opportunities for Selective Drug Development.

Medical device associated thrombosis is an important clinical problem. This type of thrombosis can result from Factor XII (FXII) binding to non-natural surface materials and subsequent activation of the contact pathway. This drives the development of new therapeutic strategies to block this pathway and information on the structural properties of FXII should catalyse this quest. Presently, there is no publicly available crystal structure of full-length FXII. However, the AlphaFold Protein Structure Database provides a model structure. We here explore this model in combination with previous structure-function studies to identify opportunities for selective pharmacological blockade of the contribution of FXII in medical device associated thrombosis. Previous studies demonstrated that FXII activation is dependent on molecular cleavage after R353. We subsequently proposed that protein conformation protects this cleavage site to ensure zymogen quiescence and prevent inappropriate FXII activation. The AlphaFold model shows that a small loop containing R353 indeed is buried in the globular molecule. This is the result of intra-molecular interactions between the (N-terminal) Fibronectin type II domain, (central) kringle and (C-terminal) protease domain, in a structure that resembles a three-point harness. Furthermore, this interaction pushes the intermediate domains, as well as the flexible proline-rich region (PRR), outward while encapsulating R353 in the molecule. The outward directed positively charged patches are likely to be involved in binding to anionic surfaces. The binding of FXII to surfaces (and several monoclonal antibodies) acccelerates its activation by inducing conformational changes. For prevention of medical device associated thrombosis, it is therefore important to target the surface binding sites of FXII without causing structural changes.

Therapeutic outcome of early-phase clinical trials in multiple myeloma: a meta-analysis.

Great progress in the treatment of patients with multiple myeloma (MM) has been made due to the development of novel drugs. Patients with relapsed/refractory MM (RRMM) can be enrolled in early-phase clinical trials, but their performance across the last decade is unknown. We conducted a meta-analysis on the overall response rate (ORR) and toxicity. PubMed, Embase, and Cochrane Library were systematically searched for phase I and phase II trials investigating an experimental compound as a single agent or in combination with dexamethasone, published from January 1, 2010 to July 1, 2020. Eighty-eight articles were included, describing 61 phase I trials involving 1835 patients and 37 phase II trials involving 2644 patients. There was a high degree of heterogeneity. Using a random-effects model, the 95% CIs of the estimated ORR were 8-17% for phase I trials and 18-28% for phase II trials. There were significant subgroup differences in ORR between the years of publication in phase I trials and between drug classes in both phase I and phase II trials. The ORR in early-phase clinical trials in RRMM is substantial, especially in phase II trials, but due to high heterogeneity a general assessment of clinical benefit before participation is difficult to offer to patients.