A thrombin receptor function for platelet glycoprotein Ib-IX unmasked by cleavage of glycoprotein V

Abstract

Glycoprotein (GP) V is a major substrate cleaved by the protease thrombin during thrombin-induced platelet activation. Previous analysis of platelets from GP V-null mice suggested a role for GP V as a negative modulator of platelet activation by thrombin. We now report the mechanism by which thrombin activates GP V -/- platelets. We show that proteolytically inactive forms of thrombin induce robust stimulatory responses in GP V null mouse platelets, via the platelet GP Ib--IX--V complex. Because proteolytically inactive thrombin can activate wild-type mouse and human platelets after treatment with thrombin to cleave GP V, this mechanism is involved in thrombin-induced platelet aggregation. Platelet activation through GP Ib-IX depends on ADP secretion, and specific inhibitors demonstrate that the recently cloned P2Y(12) ADP receptor (G(i)-coupled ADP receptor) is involved in this pathway, and that the P2Y(1) receptor (G(q)-coupled ADP receptor) may play a less significant role. Thrombosis was generated in GP V null mice only in response to catalytically inactive thrombin, whereas thrombosis occurred in both genotypes (wild type and GP V null) in response to active thrombin. These data support a thrombin receptor function for the platelet membrane GP Ib--IX--V complex, and describe a novel thrombin signaling mechanism involving an initiating proteolytic event followed by stimulation of the GP Ib--IX via thrombin acting as a ligand, resulting in platelet activation.

Figure 1

DIP-thrombin-induced aggregation in washed mouse platelets. (A). Pooled WP from GP V null mice (line 1) or wt mice (line 2) were aggregated with DIP-thrombin (100 nM). The data are the average of duplicate experiments. (B). Pooled WP from wt mice (line 1) were pretreated with thrombin as described, and aggregation was initiated by the addition of DIP-thrombin (100 nM). Line 2 shows the response of wt platelets not pretreated with thrombin to DIP-thrombin with 40 pM thrombin added simultaneously. (Inset) GP V f1 is released from wt platelets treated with 50 pM thrombin. Lanes 1 and 2 are the supernatants of control platelets, and lanes 3 and 4 are the supernatants of 50 pM thrombin-treated platelets. Lanes 1 and 3 are the immunoprecipitates with control IgG, and lanes 2 and 4 are the immunoprecipitates with Ab 808. The arrow shows the position of GP V f1. (C) GPV null platelets were aggregated with S205A-thrombin (1 μM) in the absence (line 1) or presence (line 2) of a αΙΙbβ3 inhibitor. The tracings represent experiments done at least three times with pooled platelets from four mice each.

Figure 2

DIP-thrombin-induced aggregation is inhibited by anti-GPIb antibody. WP from GP V null mice were preincubated with either anti-GPIb–IX Ab 3584 (2 μM, line 2) or control rabbit IgG (2 μM, line 1) for 10 min at room temperature. (A) Aggregation was initiated by the addition of DIP-thrombin (100 nM). (B) Aggregation was initiated by the addition of thrombin (10 nM). (C) Glycocalicin-purified Ab 3584 IgG (1.89 μM) was incubated with GP V null platelets for 10 min before the initiation of aggregation with 500 nM DIP-thrombin (line 2). Control (line 1) shows the aggregation response with 500 nM DIP-thrombin after incubation with control rabbit IgG. (D) GP V −/− WP were preincubated with either control IgG (2 μM, line 1) or Ab 3584 (2 μM, line 2) for 10 min. Aggregation was initiated by the addition of 1 μM S205A-thrombin.

Figure 3

Effect of various inhibitors on aggregation. Pooled WP were treated with the indicated concentrations of unfractionated heparin (0.3 unit/ml, A–D), or PGI₂ (4.45 μM, E–H), and aggregation responses were measured compared with that obtained in the absence of heparin or PGI₂. GP V null WP aggregated with 500 nM DIP-thrombin (A, E) or 10 nM thrombin (B, F) and thrombin-pretreated wt platelets aggregated with 500 nM DIP-thrombin (C, G) or wt platelets aggregated with 10 nM thrombin (D, H). Line 1 in each tracing represents the untreated control platelets and line 2 represents the platelets treated with inhibitor. The tracings are typical of results from experiments performed at least three times with pooled platelets from four mice each.

Figure 4

Effect of ADP receptor antagonists on DIP-thrombin-induced aggregation. (A) Pooled WP from GP V null mice were aggregated with DIP-thrombin (400 nM) in the presence (line 2) or absence (line 1) of 83 nM 2-methylthioadenosine monophosphate. (B) Pooled WP from GP V null mice were aggregated with DIP-thrombin (400 nM) in the presence (line 2) or absence (line 1) of 2 μM adenosine 3′,5′-bis-phosphate. (C) Human WP were preincubated with DMSO (line 1) or PAR 1 antagonist RWJ 56110 (40 μM; line 2) and pretreated with low doses of α-thrombin as described in Methods. Aggregation was initiated by the addition of DIP-thrombin (0.4 μM). (D) Human WP were preincubated with DMSO (line 1) or PAR 1 antagonist RWJ 56110 (40 μM; line 2). Aggregation was initiated by the addition of thrombin (1 nM).

Figure 5

Thrombosis in wt and GP V null mice. The mice were injected with 100 nM DIP-thrombin (A), 1 nM thrombin (B), 10 nM thrombin (C), 0.46 μM CHO-expressed wt DIP-thrombin (D), 0.75 μM CHO-expressed DIP-R89/R93/E94 thrombin (exosite II mutant, E) or 0.75 μM CHO-expressed DIP-R98A-thrombin (exosite II mutant, F). The number of animals used and the statistical significance is shown in each graph.

Figure 6

Thrombin-induced platelet activation. Two pathways exist on the platelet for activation by thrombin. The pathway described in this article involves the initial cleavage of GP V from the GP Ib–IX–V complex at low thrombin concentrations, resulting in a hyperresponsive platelet. Occupancy of the binding site on GP Ibα by thrombin results in a signaling response that leads to αΙΙbβ3 activation. Additionally, thrombin can cleave the PARs on platelets, and this cleavage will then also stimulate platelet aggregation.