Aβ delays fibrin clot lysis by altering fibrin structure and attenuating plasminogen binding to fibrin

Abstract

Alzheimer disease is characterized by the presence of increased levels of the ␤-amyloid peptide (A␤) in the brain parenchyma and cerebral blood vessels. This accumulated A␤ can bind to fibrin(ogen) and render fibrin clots more resistant to degradation. Here, we demonstrate that A␤ 42 specifically binds to fibrin and induces a tighter fibrin network characterized by thinner fibers and increased resistance to lysis. However, A␤ 42 -induced structural changes cannot be the sole mechanism of delayed lysis because A␤ overlaid on normal preformed clots also binds to fibrin and delays lysis without altering clot structure. In this regard, we show that A␤ interferes with the binding of plasminogen to fibrin, which could impair plasmin generation and fibrin degradation. Indeed, plasmin generation by tissue plasminogen activator (tPA), but not streptokinase, is slowed in fibrin clots containing A␤ 42 , and clot lysis by plasmin, but not trypsin, is IntroductionCerebrovascular dysfunction has been implicated as an early event in Alzheimer disease (AD) progression, 1-4 but the origins and mechanisms of vascular dysfunction in AD are not clear. The ␤-amyloid peptide (A␤) accumulates in the brain parenchyma and blood vessel walls of AD patients and has been genetically and clinically linked to AD. We have previously shown that fibrinogen, the main protein component of blood clots, can bind A␤ 42 (henceforth designated A␤) specifically with a K d of 26.3 Ϯ 6.7nM. 5 We also found that fibrin clots formed in the presence of A␤ are structurally altered and more resistant to fibrinolysis than normal clots. 6 However, the mechanism by which A␤-fibrin(ogen) binding delays fibrin clot lysis has not been defined.Fibrin clot lysis is mediated by plasmin, a serine protease that cleaves the fibrin network at specific sites. Plasmin is derived from plasminogen by tissue plasminogen activator (tPA) in the presence of fibrin, which itself enhances the rate of the reaction. One fibrin site initially involved in plasminogen activation by tPA includes residues 148-160 on the A␣-chain (reviewed by Medved and Nieuwewnhuizen 7 ). This site becomes exposed and available for plasminogen binding after the conversion of fibrinogen to fibrin, 8 but could remain hidden if clots are formed in the presence of A␤, leading to delayed clot lysis. This hypothesis is derived from our finding that A␤ binds the fibrinogen ␤-chain near the ␤-hole, 5 which is in close spatial proximity to residues 148-160 of the A␣-chain. 9 Another potential explanation for delayed clot lysis is based on the relationship between fibrin structure and its susceptibility to fibrinolysis. Tighter fibrin networks composed of thin fibers are degraded less efficiently by plasmin than those composed of thick fibers 10-13 because: (1) there are more fibers to be cleaved, 11,14 requiring plasmin to detach from and move between fibers more frequently 15 ; and (2) decreased network porosity of tighter fibrin networks results in impeded diffusion of fibrinolytic enzymes throughout the clot (...

Plasma Fibrinogen Is a Natural Deterrent to Amyloid β-Induced Platelet Activation and Neuronal Toxicity

“…Platelet-rich plasma (PRP) or washed human platelets (with or without fibrinogen) were stirred (1,200 rpm) at 37°C in an optical lumi-aggregometer (Chrono-log model 700-2, Wheecon Instruments) for 1 min, after which collagen (10 μg/mL) or different concentrations of Aβ [25][26][27][28][29][30][31][32][33][34][35] were added, and transmittance was recorded. Aggregation was measured as percentage change in light transmission, where 100% refers to transmittance through blank solution for washed platelets or plateletpoor plasma for PRP (31).…”

“…After 24 h, cells (which had reached ~80% confluence) were washed three times with a culture medium and thereafter incubated for 48 h in DMEM/F12 with 1% serum in the presence of Aβ 1-42 or Aβ [25][26][27][28][29][30][31][32][33][34][35] . In the experiments designed to examine the protective effect of fibrinogen, cells were treated with either fibrinogen or BSA (as negative control) before exposure to Aβ 1-42 or Aβ [25][26][27][28][29][30][31][32][33][34][35] . We performed the experiments using cells that had undergone fewer than 15 passages, and all studies were repeated several times with different batches of cells.…”

“…Platelets contribute to 95% of circulating amyloid precursor protein that releases Aβ into circulation. We have recently demonstrated that the Aβ active fragment containing amino acid sequence 25-35 (Aβ [25][26][27][28][29][30][31][32][33][34][35] ) is highly thrombogenic in nature and elicits strong aggregation of washed human platelets in a RhoA-dependent manner. In this study, we evaluated the influence of fibrinogen on Aβ-induced platelet activation.…”

“…Intriguingly, Aβ failed to induce aggregation of platelets suspended in plasma but not in buffer. Fibrinogen brought about dose-dependent decline in aggregatory response of washed human platelets elicited by Aβ [25][26][27][28][29][30][31][32][33][34][35] , which could be reversed by increasing doses of Aβ. Fibrinogen also attenuated Aβ-induced platelet responses such as secretion, clot retraction, rise in cytosolic Ca +2 and reactive oxygen species.…”

Pathogenic Angiogenic Mechanisms in Alzheimer's Disease

“…This establishes a cycle of neurotoxicity and death, instituted by the discharge of thrombin following Aβ-induced neuroinflammatory responses [60]. Other studies further support the interaction of Aβ with thrombin and fibrin throughout the clotting cascade, to increase neurovascular damage and neuroinflammation [61][62][63]. Astrocytes, cultured in vitro and stimulated with Aβ, showed a release of neuroinflammatory cytokines that resulted in the increased expression of VEGF [49,50].…”