Thrombin-induced conversion of fibrinogen to fibrin results in rapid platelet trapping which is not dependent on platelet activation or GPIb

Abstract

1. Activation of human platelets by thrombin is mediated by the proteolytic cleavage of two G-protein coupled protease-activated receptors, PAR-1 and PAR-4. However, thrombin also binds specifically to the platelet surface glycoprotein GPIb. It has been claimed that thrombin can induce aggregation of platelets via a novel GPIb-mediated pathway, which is independent of PAR activation and fibrinogen binding to alpha(IIb)beta(3) integrin, but dependent upon polymerizing fibrin and the generation of intracellular signals. 2. In the presence of both fibrinogen and the alpha(IIb)beta(3) receptor antagonist lotrafiban, thrombin induced a biphasic platelet aggregation response. The initial primary response was small but consistent and associated with the release of platelet granules. The delayed secondary response was more substantial and was abolished by the fibrin polymerization blocking peptide GPRP. 3. Cleavage of the extracellular portion of GPIb by mocarhagin partially inhibited thrombin-induced alpha(IIb)beta(3)-dependent aggregation and release, but had no effect on the secondary fibrin-dependent response. 4. Fixing of the platelets abolished alpha(IIb)beta(3)-dependent aggregation and release of adenine nucleotides, whereas the fibrin-dependent response remained, indicating that platelet activation and intracellular signalling are not necessary for this secondary 'aggregation'. 5. In conclusion, the secondary fibrin-dependent 'aggregation' response observed in the presence of fibrinogen and lotrafiban is a platelet trapping phenomenon dependent primarily on the conversion of soluble fibrinogen to polymerizing fibrin by thrombin.

Figure 1

Effect of fibrinogen (FIB, 0.2 mg ml⁻¹) and lotrafiban (LOT, 10 μM) on thrombin and TRAP-induced platelet aggregation and release of adenine nucleotides, ATP and ADP, in washed human platelets. (a) Thrombin concentration-response curves for rate of aggregation. Rate of aggregation is measured in arbitrary units. Fibrinogen increases the rate of aggregation, whereas lotrafiban substantially inhibits the response. The residual response in the presence of lotrafiban is due to release of platelet granules. A combination of fibrinogen and lotrafiban cause a rightward shift in the main response which is caused by the thrombin-induced polymerization of fibrin. (b) Thrombin concentration-response curves for release of ATP and ADP. Fibrinogen and lotrafiban have no effect on the thrombin-induced release of adenine nucleotides. The data points in (a) and (b) are the mean±s.e.mean, and the curves represent the best fit model, for all data from three separate experiments. (c) and (d) Trace recordings showing the effect of fibrinogen and lotrafiban on the aggregation response induced by 0.3 u ml⁻¹ thrombin and by 50 μM TRAP. Figures show the total concentration of released ATP and ADP (μM) (mean±s.e.mean). TRAP fails to induce a secondary response (measured up to 6 min) in the presence of both fibrinogen and lotrafiban, in contrast to thrombin.

Figure 2

The effect of fibrin polymerization blocking peptide GPRP (1 mM) on thrombin-induced responses in washed human platelets. (a) In the presence of fibrinogen (0.2 mg ml⁻¹) and lotrafiban (10 μM), GPRP inhibited both the rate and extent of the thrombin-induced aggregation response by preventing the polymerization of fibrin monomers, whereas it had no effect on the release of ATP and ADP. The residual response in the presence of GPRP represents the small primary response induced by thrombin which is caused by the release of platelet granules. Data shown are the mean±s.e.mean of the response to 1 u ml⁻¹ thrombin from three different experiments. (b) These traces illustrate the inhibitory effect of GPRP on the secondary thrombin-induced response in the presence of fibrinogen (0.2 mg ml⁻¹) and lotrafiban (10 μM). The smaller primary response is unaffected by GPRP. Concentrations of thrombin are shown and illustrate the concentration dependence of the delay to the onset of the fibrin-dependent secondary response. The traces are representative of three separate experiments.

Figure 3

The effect of mocarhagin on the rate of aggregation and release of ATP and ADP induced by thrombin in the absence and presence of fibrinogen (FIB, 0.2 mg ml⁻¹) and lotrafiban (LOT, 10 μM). Mocarhagin (10 μg ml⁻¹) inhibited the response to thrombin in all cases, except the aggregation response in the presence of fibrinogen and lotrafiban which is dependent on the fibrin polymerizing activity of thrombin. Data are the mean of two different experiments.

Figure 4

The effect of mocarhagin (10 μg ml⁻¹) and fixation on responses to thrombin. The control trace shows the primary and secondary responses induced by thrombin in the presence of lotrafiban (LOT, 10 μM) and fibrinogen (FIB, 0.2 mg ml⁻¹). Pre-treatment with mocarhagin substantially inhibited the primary response but did not affect the secondary fibrin-dependent response. Fixation of the platelets with formaldehyde (0.1%) and EDTA (3 mM) abolished the response to thrombin in the absence of fibrinogen. However, in the presence of fibrinogen, although there was no primary response, as seen in control platelets, the secondary response was virtually unaffected. The concentration of thrombin used was 0.3 u ml⁻¹ in all cases except for the fixed trace in which it was 1 u ml⁻¹. Traces shown are representative of those obtained from two separate experiments.

Figure 5

The effect of fixation on the response of platelets to thrombin. The fibrin polymerization-mediated response in normal platelets in the presence of fibrinogen (0.2 mg ml⁻¹) and lotrafiban (10 μM) is shown. Fixation of the platelets abolishes all responsiveness of the platelets. However, in the presence of fibrinogen, the fixed platelets manifest a rapid aggregation response. This was abolished by GPRP (1 mM), slightly attenuated by lotrafiban (10 μM), and augmented following treatment with mocarhagin (10 μg ml⁻¹). The concentration of thrombin used is 1 u ml⁻¹ in all cases. Data shown are mean±s.e.mean of three separate experiments, except for the data with mocarhagin which is from two experiments.

Figure 6

The effect on the platelet count of thrombin treatment in the presence of lotrafiban (10 μM). Thrombin-stimulated platelets from three separate experiments were fixed and the platelet count subsequently determined using a Coulter counter. For experiments 1 and 2 there was only a small reduction in platelet count at the highest concentrations of thrombin. In experiment 3, the platelet count was more substantially reduced, although, as indicated by the response to 0.03 u ml⁻¹ thrombin, there was not a consistent relationship between this effect and the small change in optical density as measured by the aggregometer. In the presence of GPRP (1 mM), the fall in the platelet count observed in experiment 3 was almost completely reversed suggesting that the micro-aggregation was predominantly mediated by the generation of small quantities of polymerizing fibrin.