Post-Translational Modifications of Platelet-Derived Amyloid Precursor Protein by Coagulation Factor XIII-A.

The physiological function of amyloid ß precursor protein (APP) in platelets has remained elusive. Upon platelet activation, APP localizes to the platelet surface and is proteolytically processed by proteases to release various metabolites, including amyloid ß (Aß) and soluble APP. Synthetic Aß is a substrate of activated coagulation factor XIII (FXIII-A*), a transglutaminase that is active both inside and on the surface of platelets. Here we tested if platelet APP and its fragments are covalently modified by FXIII-A*. Platelet-derived FXIII-A* and fibrin(ogen) bound to APP, and their bound fractions increased 7- and 11-fold upon platelet activation, respectively. The processing of platelet APP was enhanced when FXIII-A* was inhibited. Soluble APPß was covalently cross-linked by FXIII-A*. This mechanism regulating APP processing is significant, because controlling the processing of APP, such as by inhibiting specific secretases that cleave APP, is a therapeutic target for Alzheimer's disease.

Bleeding is increased in amyloid precursor protein knockout mouse.

BACKGROUND: Amyloid precursor protein (APP) is highly expressed in platelets. APP is the precursor to amyloid beta (Aß) peptides that accumulate in cerebral amyloid angiopathy and plaques in Alzheimer disease. APP and its metabolites interact with many components of the coagulation system, and have both anticoagulant and procoagulant properties, but it is unclear if APP contributes to hemostasis in vivo. OBJECTIVES: To determine whether APP contributes to hemostasis in mice, including when inhibitors of coagulation are administered. METHODS: Blood loss in APP knockout (KO) mice was measured in liver laceration and tail transection models of hemorrhage. Blood loss was also measured following tail transection in mice given an inhibitor of coagulation factor Xa (apixaban), platelet inhibitors (aspirin + clopidogrel), tissue-type plasminogen activator (t-PA), or the antifibrinolytic tranexamic acid (TXA). RESULTS AND DISCUSSION: Blood loss from liver lacerations was similar between APP KO mice and wild-type (WT) mice, but APP KO mice bled more from tail transections. When mice were challenged with aspirin + clopidogrel, the difference in bleeding between APP KO and WT mice was abrogated. In contrast, a difference in bleeding between the strains persisted when mice were treated with apixaban, t-PA, or TXA. Blood collected from APP KO mice and analyzed with thromboelastography had longer clotting times, and the clots were less stiff and more susceptible to fibrinolysis compared to blood from WT mice. CONCLUSIONS: The absence of APP measurably increases bleeding in mice, which is consistent with a role for platelet-derived APP and Aß peptides in hemostasis.

Coagulation factor XII contributes to hemostasis when activated by soil in wounds.

Bleeding is a common contributor to death and morbidity in animals and provides strong selective pressure for the coagulation system to optimize hemostasis for diverse environments. Although coagulation factor XII (FXII) is activated by nonbiologic surfaces, such as silicates, which leads to blood clotting in vitro, it is unclear whether FXII contributes to hemostasis in vivo. Humans and mice lacking FXII do not appear to bleed more from clean wounds than their counterparts with normal FXII levels. We tested the hypothesis that soil, a silicate-rich material abundant in the environment and wounds of terrestrial mammals, is a normal and potent activator of FXII and coagulation. Blood loss was compared between wild-type (WT) and FXII-knocked out (FXII-/-) mice after soil or exogenous tissue factor was applied to transected tails. The activation of FXII and other components of the coagulation and contact system was assessed with in vitro coagulation and enzyme assays. Soils were analyzed by time-of-flight secondary ionization mass spectrometry and dynamic light scattering. Soil reduced blood loss in WT mice, but not FXII-/- mice. Soil accelerated clotting of blood plasma from humans and mice in a FXII-dependent manner, but not plasma from a cetacean or a bird, which lack FXII. The procoagulant activity of 13 soils strongly correlated with the surface concentration of silicon, but only moderately correlated with the ζ potential. FXII augments coagulation in soil-contaminated wounds of terrestrial mammals, perhaps explaining why this protein has a seemingly minor role in hemostasis in clean wounds.

Coagulation factor XIIIa cross-links amyloid ß into dimers and oligomers and to blood proteins.

In cerebral amyloid angiopathy (CAA) and Alzheimer's disease (AD), the amyloid ß (Aß) peptide deposits along the vascular lumen, leading to degeneration and dysfunction of surrounding tissues. Activated coagulation factor XIIIa (FXIIIa) covalently cross-links proteins in blood and vasculature, such as in blood clots and on the extracellular matrix. Although FXIIIa co-localizes with Aß in CAA, the ability of FXIIIa to cross-link Aß has not been demonstrated. Using Western blotting, kinetic assays, and microfluidic analyses, we show that FXIIIa covalently cross-links Aß40 into dimers and oligomers (kcat/Km = 1.5 × 105 m-1s-1), as well as to fibrin, platelet proteins, and blood clots under flow in vitro Aß40 also increased the stiffness of platelet-rich plasma clots in the presence of FXIIIa. These results suggest that FXIIIa-mediated cross-linking may contribute to the formation of Aß deposits in CAA and Alzheimer's disease.

Localization of Short-Chain Polyphosphate Enhances its Ability to Clot Flowing Blood Plasma.

Short-chain polyphosphate (polyP) is released from platelets upon platelet activation, but it is not clear if it contributes to thrombosis. PolyP has increased propensity to clot blood with increased polymer length and when localized onto particles, but it is unknown whether spatial localization of short-chain polyP can accelerate clotting of flowing blood. Here, numerical simulations predicted the effect of localization of polyP on clotting under flow, and this was tested in vitro using microfluidics. Synthetic polyP was more effective at triggering clotting of flowing blood plasma when localized on a surface than when solubilized in solution or when localized as nanoparticles, accelerating clotting at 10-200 fold lower concentrations, particularly at low to sub-physiological shear rates typical of where thrombosis occurs in large veins or valves. Thus, sub-micromolar concentrations of short-chain polyP can accelerate clotting of flowing blood plasma under flow at low to sub-physiological shear rates. However, a physiological mechanism for the localization of polyP to platelet or vascular surfaces remains unknown.

Coagulation factor XIIIa is inactivated by plasmin.

Coagulation factor XIIIa (FXIIIa) is a transglutaminase that covalently cross-links fibrin and other proteins to fibrin to stabilize blood clots and reduce blood loss. A clear mechanism to describe the physiological inactivation of FXIIIa has been elusive. Here, we show that plasmin can cleave FXIIIa in purified systems and in blood. Whereas zymogen FXIII was not readily cleaved by plasmin, FXIIIa was rapidly cleaved and inactivated by plasmin in solution (catalytic efficiency = 8.3 × 10(3) M(-1)s(-1)). The primary cleavage site identified by mass spectrometry was between K468 and Q469. Both plasma- and platelet-derived FXIIIa were susceptible to plasmin-mediated degradation. Inactivation of FXIIIa occurred during clot lysis and was enhanced both in plasma deficient in fibrinogen and in plasma treated with therapeutic levels of tissue plasminogen activator. These results indicate that FXIIIa activity can be modulated by fibrinolytic enzymes, and suggest that changes in fibrinolytic activity may influence cross-linking of blood proteins.