The GPIb-thrombin pathway: evidence for a novel role of fibrin in platelet aggregation.

It has long been presumed that fibrinogen and its thrombin proteolytic product, fibrin, are the crosslinking agents that bind to the glycoprotein IIb/IIIa (GPIIb/IIIa) receptor in aggregating platelets. We reported that a-thrombin could induce human platelet aggregation via a fibrin-dependent GPIba pathway [1], others demonstrated that this pathway depends on ADP secretion in a mouse system [2]. The interpretation of our studies has been questioned by Jarvis and coworkers [3]. However, their washed platelet samples contained 800 nM prostacyclin (PGI2), a platelet inhibitor [4], which they let rest for 1 h to inactivate PGI2. The PGI2 is not inactivated in 1 h and continues to suppress aggregation (Table 1). Jarvis et al. claimed that our observations were due to fibrin trapping of platelets. The reason Jarvis et al. saw no aggregation was probably due to the insensitivity of their preparations to ADP. A spatial and functional interplay between the glycoprotein Ib–IX–V complex and the FccRIIA receptor has been reported [5,6]. We found that protease activated receptor-1 (PAR-1) inhibited (with SCH203099), and PAR-4 desensitized platelets still aggregate in response to a-thrombin via the GPIb pathway [1]. If these platelets are also pretreatedwith the fab fragment of the antibody LJIb-10 (binds to the thrombin binding region of GPIb [7]) or with the antibody IV.3, specific for the FccRIIA receptor, their ability to aggregate is diminished (Fig. 1Ab,Ac). If LJIb-10 and IV.3 are added together, inhibition is nearly complete (Fig. 1Ad). Finally, the anti1433 protein antibody (used for other experiments) totally blocked a-thrombininduced aggregation (Fig. 1Bb). It is likely that the antibodies bound to the FccRIIA receptor sterically block a-thrombin binding to GPIba. In each case, a-thrombin would still hydrolyze the added 100 lg mL fibrinogen to fibrin, yet we detect no trapping. Two reports demonstrate that two molecules of a-thrombin bind to a single GPIbamolecule and may help explain what we have observed as a fibrin-dependent aggregation pathway. One thrombin molecule binds through its exosite I while the second binds through exosite II [8,9], which would account for the GPIb activation of platelets [1], the release of ADP to reinforce aggregation [2], and the binding of generated fibrin to the GPIba-bound thrombin that we demonstrate here. a-Thrombin binds to fibrin [10,11]. Our studies indicate that fibrin could crosslink platelets via exposed exosite I regions on the athrombin–GPIba complexes between platelets and thus form aggregates (Fig. 1), independent of fibrinogen binding to GPIIb/IIIa. A similar ternary complex has been proposed for fibrin–a-thrombin–heparin, where a-thrombin binds to fibrin through exosite I and heparin binds through exosite II [11]. As fibrin polymerizes it would build upon the initial fibrin molecules that crosslink platelets via the bound thrombin. This latter phase may mimic a trapping mechanism but would, in fact, be dependent upon the underlying specific fibrindependent aggregation and ADP–platelet interactions. Others have shown that some event other than fibrinogen binding is critical for ADP-induced platelet aggregation [12,13]. The initiation or inhibition of platelet aggregations under a variety of conditions were essentially the same with platelets that were untreated or treated with 7.5 lM SCH203099, a specific PAR-1 inhibitor (Fig. 1D–J), indicating that PAR-1 does not influence the response. Endogenous and/or surfacebound fibrinogen is not present in sufficient quantities to support aggregation in the presence of 230 lM RGDS, a peptide that competes with fibrinogen binding to GPIIb/IIIa (Fig. 1E). This inhibition is overcome, after a lag period, by the addition of 100 lg mL fibrinogen (Fig. IF). The lag time represents the period required for the a-thrombin generation of fibrin monomers/polymers. This lag time is reduced by first preincubating the a-thrombin with fibrinogen for 2 min followed by the addition of platelets. The added RGDS has little effect on the subsequent aggregation response (Fig. 1G). The aggregation observed in Fig. 1G is not due to fibrin trapping but requires a feedback mechanism by released ADP since the ADP hydrolyzing enzyme, apyrase, abrogates all but the initial response (Fig. 1H). The fact that the aggregation detected in Fig. 1F,G is not due to fibrin trapping is further Correspondence: Gerald Soslau, Department of Biochemistry, Drexel University College of Medicine, Philadelphia, PA, USA. Tel.: 215 762 7831; fax: 215 762 7434; e-mail: gerald.soslau@drexel. edu