Abstract
Factor XII deficiency has been postulated to be a risk factor for thrombosis suggesting that factor XII is an antithrombotic protein. The biochemical mechanism leading to this clinical observation is unknown. We have previously reported high molecular weight kininogen (HK) inhibition of thrombin-induced platelet aggregation by binding to the platelet glycoprotein (GP) Ib-IX-V complex. Although factor XII will bind to the intact platelet through GP Ibalpha (glycocalicin) without activation, we now report that factor XIIa (0. 37 microm), but not factor XII zymogen, is required for the inhibition of thrombin-induced platelet aggregation. Factor XIIa had no significant effect on SFLLRN-induced platelet aggregation. Moreover, an antibody to the thrombin site on protease-activated receptor-1 failed to block factor XII binding to platelets. Inhibition of thrombin-induced platelet aggregation was demonstrated with factor XIIa but not with factor XII zymogen or factor XIIf, indicating that the conformational exposure of the heavy chain following proteolytic activation is required for inhibition. However, inactivation of the catalytic activity of factor XIIa did not affect the inhibition of thrombin-induced platelet aggregation. Factor XII showed displacement of biotin-labeled HK (30 nm) binding to gel-filtered platelets and, at concentrations of 50 nm, was able to block 50% of the HK binding, suggesting involvement of the GP Ib complex. Antibodies to GP Ib and GP IX, which inhibited HK binding to platelets, did not block factor XII binding. However, using a biosensor, which monitors protein-protein interactions, both HK and factor XII bind to GP Ibalpha. Factor XII may serve to regulate thrombin binding to the GP Ib receptor by co-localizing with HK, to control the extent of platelet aggregation in vivo.
Highlights
The functions of the contact system have been reevaluated in view of our increasing knowledge of the interactions of factor XII, prekallikrein, and kininogens with platelets, neutrophils, monocytes, and endothelial cells
Inhibition of thrombin-induced platelet aggregation was demonstrated with factor XIIa but not with factor XII zymogen or factor XIIf, indicating that the conformational exposure of the heavy chain following proteolytic activation is required for inhibition
The light chain of factor XIIa is homologous to plasmin, an enzyme that participates in fibrinolysis, and to tissue plasminogen activator, a protease that converts plasminogen to plasmin in the fibrinolytic pathway
Summary
The functions of the contact system have been reevaluated in view of our increasing knowledge of the interactions of factor XII, prekallikrein, and kininogens with platelets, neutrophils, monocytes, and endothelial cells. The binding site on platelets, which mediates this effect, was shown to be GP Ib-IX complex [1] This observation helps to explain the fact that patients with BernardSoulier disease require 10-fold the concentration of thrombin needed to stimulate normal platelets [6]. Kininogens shift the dose-response curve for thrombin activation of platelets about 10-fold [1, 4] These studies were confirmed by Joseph et al [7], who demonstrated that a molecule of 74 kDa, isolated by ligand affinity chromatography utilizing HK, was a fragment of the GP Ib␣ chain. A third human protein, PAR4 [12], has been shown to be involved in thrombin activation of platelets at high thrombin concentrations, e.g. 30 nM [13]. The heavy chain contains a proline-rich region making up 33% of the sequence, the significance of which is unclear at this time, but it most likely functions as a connection domain between the light chain and heavy chains of factor XII
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