Elsevier

Thrombosis Research

Volume 131, Issue 4, April 2013, Pages 338-341
Thrombosis Research

Regular Article
Factor XIII improves platelet adhesion to fibrinogen by protein disulfide isomerase-mediated activity

https://doi.org/10.1016/j.thromres.2012.12.003Get rights and content

Abstract

Background

Factor XIII (FXIII), a plasma pro-transglutaminase, consists of two A subunits and two B subunits (FXIIIA2B2). Following activation by thrombin, it cross-links fibrin chains at the final step of coagulation. We previously reported that FXIII subunit A (FXIIIA) serves as a protein disulfide isomerase (PDI), and that PDI promotes platelet adhesion and aggregation.

Objective

This study sought to examine possible mechanistic effect of FXIII on platelet adhesion to fibrinogen; specifically, the role of its PDI activity.

Methods

Ex vivo experiments: Blood platelets derived from five patients with hereditary FXIIIA deficiency before and after treatment with Fibrogammin-P (FXIIIA2B2 concentrate) were washed and incubated on immobilized fibrinogen. Bound platelets were stained and counted by microscopy. In vitro experiments: Platelets derived from patients before treatment and five healthy controls were washed and analyzed for adhesion in the presence or absence of Fibrogammin-P or recombinant FXIII (FXIIIA2 concentrate).

Results

In ex vivo experiments, one hour after Fibrogammin-P treatment, mean (± SEM) platelet adhesion to fibrinogen increased by 27 ± 2.32% (p < 0.001). In in vitro experiments, treatment with Fibrogammin-P or recombinant FXIII (10 IU/mL each) enhanced platelet adhesion to fibrinogen (in patients, by 29.95 ± 6.7% and 29.05 ± 5.3%, respectively; in controls, by 26.06 ± 3.24% and 26.91 ± 4.72, respectively; p < 0.04 for all). Iodoacetamide-treated FXIII (I-FXIII), where transglutaminase activity is blocked, showed similar enhanced adhesion as untreated FXIII. By contrast, addition of an antibody that specifically blocks FXIIIA-PDI activity inhibited FXIII-mediated platelet adhesion to fibrinogen by 65%.

Conclusion

These findings indicate that FXIII-induced enhancement of platelet adhesion is mediated by FXIII-PDI activity.

Introduction

Coagulation factor XIII (FXIII) is a plasma pro-transglutaminase. Following activation by thrombin, it cross-links fibrin chains at the final step of coagulation to form a soluble clot [1]. FXIII circulates in plasma as a heterotetramer of two A subunits (FXIIIA2 - the active transglutaminase) and two B subunits (FXIIIB2 – the carrier protein) [1]. Besides its role in hemostasis, FXIII accelerates wound healing [2], probably by its pro-angiogenic effects [3] as well as by stimulation of monocytes and fibroblasts [4]. Several new lines of evidence point to the involvement of FXIII in platelet function as well [5], [6], [7], [8]. FXIII subunit A (FXIIIA) was detected on thrombin-receptor-activated platelets [5], and platelets were found to adhere to FXIII-covered surface [6]; this interaction depended on the intact fibrinogen binding to integrin αIIbβ3 [5], [6]. Accordingly, no clot retraction was noted in FXIIIA knock-out mice [7], and patients with FXIII deficiency, a rare hereditary life-long bleeding disorder [8], showed reduced fibrinogen binding to thrombin-stimulated platelets and reduced platelet adhesion to fibrinogen-covered surface [9].

We recently reported that FXIIIA has protein disulfide isomerase (PDI) activity, independent of its transglutaminase activity [10]. Cell-surface-localized, membrane-bound PDI is essential for sustaining the binding of fibrinogen, fibronectin, and collagen to platelet integrins αIIbβ3, α5β1, and α2β1, respectively [11], [12], thereby regulating platelet adhesion [11], [12] and aggregation [13]. PDI also regulates the activity of L-selectin, a cell-adhesion molecule found on leukocytes [14]. Specifically, it was found to mediate the entry of human immunodeficiency virus into lymphocytes [15] and the entry of nitric oxide into cells [16].

In the present study, on the basis of our earlier findings that PDI plays a mediatory role in platelet adhesion [11], [12], [13] and that FXIIIA exerts PDI activity [10], we sought to examine the relative contributions of FXIII PDI or transglutaminase activity to platelet adhesion to fibrinogen.

Section snippets

Materials

Fibrogammin-P (FXIIIA2B2 concentrate) was purchased from ZLB-Behring, Marburg, Germany. Recombinant FXIII (rFXIII) was a gift from Novo Nordisk, Bagsvaerd, Denmark. Purified FXIII, used to test the inhibitory effect of anti-FXIII antibody, was described previously [10]. Rabbit anti-human factor XIII A-subunit antiserum, (Assera XIII A), was purchased from Diagnostica Stago, Asnieres sur Seine, France. Sheep anti-human factor XIIIA2B2 (SAXIII-IG) was purchased from Affinity Biologicals Inc.

Results

The ex vivo findings shown in Fig. 1 represent the summation of 56 treatments in five patients with hereditary FXIIIA deficiency. One hour after Fibrogammin-P infusion, platelet adhesion to fibrinogen increased by a 27 ± 2.32% from the pretreatment level (p < 0.001).

Results of the in vitro experiments are presented in Fig. 2. The addition of Fibrogammin-P (10 IU/mL) to the washed platelets from the patients enhanced adhesion to fibrinogen by 29.95 ± 6.7%, and the addition of rFXIII (10 1U/mL) enhanced

Discussion

The involvement of FXIII transglutaminase activity in platelet spreading was suggested by previous findings that monodansylcadaverine, in the absence of added FXIII, diminished filopodia formation of normal platelets on fibrinogen [9] and that clot retraction in mice was inhibited by the FXIII transglutaminase inhibitor, cystamine [7]. Given our earlier finding that FXIIIA has PDI activity which is independent of its transglutaminase activity [10], we examined the relative roles of the two

Conflict of interest statement

None of the authors has a direct or indirect proprietary interest in the manuscript.

Funding source

This work was supported in part by grants from the Chief Scientist's Office of the Ministry of Health, Israel.

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