Abstract
Thrombin activation of platelets induces the release of a high molecular weight glycoprotein, thrombospondin. On treatment with factor XIII transglutaminase and [3H]putrescine, thrombospondin undergoes specific incorporation of this labeled amine, with 2-3 mol of putrescine being incorporated per mol of thrombospondin. Analysis of plasmin digests of [3H]putrescine-thrombospondin showed that the Mr 53,000-core peptide contains the glutamine site for amine incorporation. In the absence of amine substrate, thrombospondin was found to provide both donor (glutamine) and acceptor (lysine) sites for intermolecular cross-links by factors XIIIa, and high molecular weight protein complexes were formed. Homopolymers of thrombospondin were also observed by electron microscopy. Thrombin-cleaved thrombospondin has more cross-linking sites accessible for [3H]putrescine incorporation or for cross-linkage to itself than does the uncleaved native protein. Examination of thrombospondin cross-linkage in the presence of other protein substrates (fibronectin, collagen, fibrinogen, and von Willebrand factor) for factor XIIIa, resulted in reduced thrombospondin polymer formation. Electron microscopy and autoradiography of fibrin clots formed in the presence of 125I-thrombospondin showed an association of thrombospondin with fibrin fibrils. However, confirmation that this association involves covalent epsilon-(gamma-glutamyl)lysyl cross-links between thrombospondin and fibrin was not obtained.
Highlights
Thrombin activation of platelets induces the release din may mediate aggregation by binding to a specific receptor of a high molecular weight glycoprotein, thrombospondin
When assayed under reducing conditions for 2 h at 37 "C, Preparation of Fibrin Clots for Electron Microscopy and Autora- with factor XIIIaandthrombin which had been inhibited diography-Blood was drawn by venipuncture from a healthy donor
Evidence is presented that theplatelet release protein thrombospondin is a substrate for amine incorporation catalyzed by factor XIIIa,and for cross-linkage with itself
Summary
Thrombin activation of platelets induces the release din may mediate aggregation by binding to a specific receptor of a high molecular weight glycoprotein, thrombospondin. The aim of the present study was to characterize thrombospondin as a substratfeor factor XIIIa. FactorXI11becomes activated during blood coagulation to factor XIIIa and catalyzes covalent cross-links in fibrin [18]and anumber of other plasma and platelet proteins (fibronectin, actin, von Willebrand factor, factor V, az-plasmininhibitor) [19,20,21,22,23,24]. FactorXI11becomes activated during blood coagulation to factor XIIIa and catalyzes covalent cross-links in fibrin [18]and anumber of other plasma and platelet proteins (fibronectin, actin, von Willebrand factor, factor V, az-plasmininhibitor) [19,20,21,22,23,24] It is din cross-linkage inthe presence of other proteinsub- shown that thrombospondin is a substrate for factor XIIIa strates
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