Key Points• PolyP significantly augments the plasminogen activator capacity of FXIIa.• Platelet-bound fibrin acts as a reservoir for plasminogen, FXII(a), and polyP.Activated factor XII (FXIIa) has plasminogen activator capacity but its relative contribution to fibrinolysis is considered marginal compared with urokinase and tissue plasminogen activator. Polyphosphate (polyP) is released from activated platelets and mediates FXII activation. Here, we investigate the contribution of polyP to the plasminogen activator function of aFXIIa. We show that both polyP 70 , of the chain length found in platelets (60-100 mer), and platelet-derived polyP significantly augment the plasminogen activation capacity of aFXIIa. PolyP 70 stimulated the autoactivation of FXII and subsequent plasminogen activation, indicating that once activated, aFXIIa remains bound to polyP 70 . Indeed, complex formation between polyP 70 and aFXIIa provides protection against autodegradation. Plasminogen activation by bFXIIa was minimal and not enhanced by polyP 70 , highlighting the importance of the anion binding site. PolyP 70 did not modulate plasmin activity but stimulated activation of Glu and Lys forms of plasminogen by aFXIIa. Accordingly, polyP 70 was found to bind to FXII, aFXIIa, and plasminogen, but not bFXIIa. Fibrin and polyP 70 acted synergistically to enhance aFXIIa-mediated plasminogen activation. The plasminogen activator activity of the aFXIIa-polyP 70 complex was modulated by C1 inhibitor and histidine-rich glycoprotein, but not plasminogen activator inhibitors 1 and 2. Platelet polyP and FXII were found to colocalize on the activated platelet membrane in a fibrin-dependent manner and decorated fibrin strands extending from platelet aggregates. We show that in the presence of platelet polyP and the downstream substrate fibrin, aFXIIa is a highly efficient and favorable plasminogen activator. Our data are the first to document a profibrinolytic function of platelet polyP. (Blood. 2016;128(24):2834-2845
Investigations were carried out into the activity and localization of NADHdependant diaphorase in boar spermatozoa. Semen samples were collected from healthy boars, used in A.I. centers. The enzyme was extracted with distilled water and Triton X-100. Two forms of diaphorase were found-water-soluble and Triton X-100 soluble, showing low activity-O,36U/mI and 0,26U/ml. The enzyme was localized in the mitochondria, manifesting different intensities of reaction between sperm cells in the same ejaculate. It was found, that a part of the mitochondria and outer doublets showed positive reaction. It is suggested that the enzyme regulates the ratio between reduced and oxidized forms of NADH, takes part in the energy balance and possibly in the mechanism of sperm motility. Aktivitat und Lokalisation von NADH-abhangiger Oxydoreduktase (Diaphorase) in EberspermatozoenZusammenfassung: Die Aktivitat der NADH-abhangigen Diaphorase wurde in Spermatozoen von Ebern untersucht. Das Ejakulat hierfiir wurde von gesunden Ebern gesammelt, die im A.I.Zentrum gehalten wurden. Die Extraktion des Enzyms erfolgte mit destilliertem Wasser und Triton X-100. Dabei ergab sich, dab zwei verschiedene Formen der Diaphorase gefunden werden konnten und zwar wasserloslich und Triton X-100 loslich, die eine niedrige Aktivitat ( -0,36U/ml und 0,26U/ml) aufwieseu. Das Enzym war in den Mitochondrien lokalisiert und bewirkte eine verschiedene Reaktionsintensitat zwischen den Spermazellen im selben Ejakulat. Es fand sich, dab ein Teil der Mitochondrien und der auSeren Hullen eine positive Reaktion zeigten. Es ist wahrscheinlich, daS das Enzym die Relation zwischen reduzierter und oxidierter Form der NADH reguliert, bei der Energiebalance und moglicherweise auch beim Mechanismus der Samenbeweglichkeit eine Rolle spielt.
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