Platelet recruitment to sites of vascular injury is mediated by von Willebrand factor (VWF). The shear-induced unraveling of ultra-large VWF multimers causes the formation of a “stringlike” conformation, which rapidly recruits platelets from the bloodstream. A disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) regulates this process by cleaving VWF to prevent aberrant platelet adhesion; it is unclear whether the activity of ADAMTS13 itself is regulated. The serine proteases α-thrombin and plasmin have been shown to cleave ADAMTS13. Based on sequence homology, we hypothesized that activated coagulation factor XI (FXIa) would likewise cleave ADAMTS13. Our results show that FXIa cleaves ADAMTS13 at the C-terminal domains, generating a truncated ADAMTS13 with a deletion of part of the thrombospondin type-1 domain and the CUB1-2 domains, while α-thrombin cleaves ADAMTS13 near the CUB1-2 domains and plasmin cleaves ADAMTS13 at the metalloprotease domain and at the C-terminal domain. Using a cell surface immunoassay, we observed that FXIa induced the deletion of the CUB1-2 domains from ADAMTS13 on the surface of endothelial cells. Removal of the C-terminal domain of ADAMTS13 by FXIa or α-thrombin caused an increase in ADAMTS13 activity as measured by a fluorogenic substrate (FRETS) and blocked the ability of ADAMTS13 to cleave VWF on the endothelial cell surface, resulting in persistence of VWF strands and causing an increase in platelet adhesion under flow conditions. We have demonstrated a novel mechanism for coagulation proteinases including FXIa in regulating ADAMTS13 activity and function. This may represent an additional hemostatic function by which FXIa promotes local platelet deposition at sites of vessel injury.
The integration of biomaterials and understanding of vascular biology has led to the development of perfusable endothelialized flow models, which have been used as valuable tools to study the platelet-endothelium interface under shear. In these models, the parameters of geometry, compliance, biorheology and cellular complexity are varied to recapitulate the physical biology of platelet recruitment and activation under physiologically relevant conditions of blood flow. In this review, we summarize the mechanistic insights learned from perfusable microvessel models and discuss the potential utility as well as challenges of endothelialized microfluidic devices to study platelet function in the bloodstream in vitro.
Background: ADAMTS13 cleaves and inactivates von Willebrand factor (VWF), which binds collagen, facilitating platelet adhesion under vascular injury. But is still uncertain how ADAMTS13 activity is regulated. Thrombin and plasmin have been shown to cleave ADAMTS13. Based on the fact that elevated levels of FXI is an independent risk factor for deep vein thrombosis and ischemic stroke, we hypothesize that FXIa inactivates ADAMTS13 leading to platelet aggregation and thrombus formation. Aim: To determine the functional role of inactivation of ADAMTS13 by FXIa. Methods and Results: Recombinant ADAMTS13 (250 nM) was incubated with FXIa (50 nM) for increasing times (0-3 hours) at 37 o C before being analyzed by western blot using an anti-ADAMTS13 antibody against the two CUB domains (C-terminal) or against the metalloproteinase (MET) domain (N-terminal). Our results show that FXIa caused the disappearance of the ADAMTS13 band (~200 kDa) and the appearance of a band at ~150 kDa when the samples were analyzed with the anti-MET antibody and a ~50 kDa band when the samples were analyzed with the anti-CUB antibody. The presence of aprotinin, which inhibits FXIa activity, blocked the degradation of ADAMTS13. Kallikrein or FXIIa were unable to cleave ADAMTS13. Using a cell surface immunoassay we observed that after incubation with FXIa, the detection of the CUB domain from ADAMTS13 was lost from endothelial cells surface. The incubation of ADAMTS13 with FXIa caused an increase in ADAMTS13 activity as measured by a fluorogenic substrate (FRETS). Conclusion: ADAMTS13 circulates in a closed conformation, which is maintained by a CUB-spacer domain binding interaction. ADAMTS13 becomes conformationally activated through interaction of its CUBs domains with VWF. Here we show that FXIa-mediated deletion of ADAMTS13-CUB domains enhances its capacity to cleave FRETS and blocks the interaction with VWF. Our results suggest that FXIa may limit ADAMTS13-mediated VWF inactivation.
Background: ADAMTS13, a plasma metalloprotease, is secreted into blood as an active enzyme that cleaves and inactivates von Willebrand factor (VWF), which binds collagen, facilitating platelet adhesion under vascular flow. Plasma ADAMTS13 has a molecular weight of 200 kDa, consisting of a metalloprotease (MET) domain, a disintegrin-like domain, a first thrombospondin type-1 repeat (TSP1) domain, a Cys-rich domain, and a spacer domain. Moreover, the C-terminal domain of ADAMTS13 contains an additional seven TSP1 repeats and two CUB domains. ADAMTS13 has been shown to adopt a natural folded conformation, allowing its CUB domains to interact with its spacer domain. This more closed conformation prohibits the functional exosite on the spacer domain from interacting with its proteolytic site on the A2 domain of VWF. In plasma, globular ADAMTS13 will associate with VWF via necessary binding of the CUB domains to the VWF D4CK fragment. Under shear stress or flow conditions, bound ADAMTS13 will unfold leading to exposure of the spacer domain exosite and ultimately increased ADAMTS13 proteolysis VWF. Without the CUB domains, ADAMTS13 does not proteolyze VWF under flow conditions. To date, it is still uncertain how ADAMTS13 activity is regulated, and what impact this has on the inactivation of VWF. The serine proteases thrombin, activated FX (FXa), and plasmin have been shown to cleave and inactivate ADAMTS13. Based on the fact that congenital factor XI deficiencies are associated with bleeding disorders and that elevated levels of FXI is an independent risk factor for deep vein thrombosis and ischemic stroke, we hypothesize that the serine protease activated FXI (FXIa) inactivates ADAMTS13 leading to platelet aggregation and thrombus formation. Aim: To determine whether FXIa is able to cleave and inactivate ADAMTS13. Methods and results: Recombinant ADAMTS13 (250 nM) was incubated with FXIa (50 nM) for selected times (0-3 hours) at 37oC before being separated by SDS-PAGE and analyzed by Coomassie blue staining, resulting in the disappearance of the ADAMTS13 band (~200 kDa) and the appearance of lower molecular weight bands under reducing conditions. The presence of aprotinin, which inhibits FXIa activity, blocked the degradation of ADAMTS13 by FXIa. Samples were analyzed by western blot to determine the cleavage site using an anti-ADAMTS13 antibody, which specifically binds the two CUB domains, and an anti-ADAMTS13 antibody which specifically binds the MET domain. ADAMTS13 has been shown to be cleaved by serine proteases, such as plasmin, thrombin and FXa. We incubated ADAMTS13 (200nM) with equivalent concentrations of plasmin, thrombin, FXa, FXIa, FXIIa or Kallikrein at 37oC over a time interval of 0-3 hours. The addition of Ca++(5 mM) was necessary for proteolytic activity of thrombin and FXa. We observed that the ability of FXIa to cleave ADAMTS13 was found to be similar to the ability of thrombin to cleave ADAMTS13. Neither FXa, kallikrein, nor FXIIa appeared to cleave ADAMTS13. The antibody against the MET domain detected a single broad band at approximately 150 kDa. When the samples were analyzed with the antibody specific for the two CUB domains, a single broad band at approximately 50 kDa was detected, suggesting that the proteolysis of ADAMTS13 by FXIa preferentially occurs near the start of the first CUB domain. Interestingly, it has been previously reported that the CUB domains are necessary for VWF strand cleavage under flow conditions. It has been shown that the cleavage of a fluorescence-quenching substrate, FRETS-VWF73, by ADAMTS13 was enhanced after CUB1-2 domain removal. We observed that after the incubation of ADAMTS13 (30nM) with FXIa (30nM) at 37oC for 3 hours, the activity of ADAMTS13 was increased. Analysis of the samples by western blot using an anti-ADAMTS13 MET antibody confirmed the generation of the 150 kDa fragment. Conclusion: Our study suggests a novel molecular link between the regulation of VWF activity and FXI through inactivation of ADAMTS13. The results suggest that the hemostatic role of FXIa may be attributed not only to activation of FIX but also through limiting ADAMTS13-mediated VWF inactivation. Our future studies are focused on determining the physiological role of the proteolytic removal of the CUB domains of ADAMTS13 by FXIa under flow conditions by measuring platelet aggregation. Disclosures No relevant conflicts of interest to declare.
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