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Congres Report
 

Mikamo Lecture

 
Protease-Activated Receptors in Hemostasis and Thrombosis
Shaun Coughlin
Cardiovascular Research Institute, University of California, San Francisco, USA
 

Dr. Shaun Coughlin, University of California San Francisco, discussed the role of PARs in hemostasis and thrombosis and the rationale for the PAR1 receptor as a target for antithrombotic therapy. The PAR1 receptor mediates cellular responses to thrombin. Positive and negative feedback and inhibitors make thrombin generation fast and local. Thrombin is a protease that cleaves fibrinogen to fibrin monomer, which polymerizes to form the fibrous meshwork that supports blood clots. Thrombin also regulates cell behavior much as a hormone does.

Figure 1. Activation of PAR1 by Thrombin.
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Dr. Coughlin’s laboratory identified the PAR1 receptor. PAR1 is cleaved by thrombin at a single site, which unmasks a new amino terminus, which serves as a tethered ligand and docks intramolecularly with the heptahelical bundle of the receptor to mediate transmembrane signaling and G protein activation (Figure 1). Thrombin can function like a hormone because of PAR1, a peptide receptor that carries its own ligand, which is unmasked by proteolysis.

There are four PARs in humans and mice. PAR1, PAR3, and PAR4 can be activated by thrombin and there is evidence that they mediate cellular responses to thrombin in vivo. In human platelets, PAR1 is required for normal responsiveness to low concentrations of thrombin. Inhibition of PAR1 leads to a right-shift in the concentration-response to thrombin. The residual platelet responses to higher thrombin concentrations seen in PAR1-inhibited human platelets are PAR4-dependent. Inhibition of both PAR1 and PAR4 eliminates responses to thrombin in human platelets.

Mice deficient in prothrombin die at birth from fatal hemorrhage. Mice lacking fibrinogen tend to bleed but have a normal lifespan, and mice deficient in PAR4 have no spontaneous bleeding. Dr. Coughlin found that fibrinogen and PAR4 double knockout mice hemorrhaged and died at birth, demonstrating the importance of thrombin for hemostasis. Importantly, even complete ablation of platelet activation by thrombin does not cause spontaneous bleeding, suggesting that PARs might be relatively safe compared to traditional anticoagulation.

Figure 2. Kinetics of Platelet Accumulation in Wild-Type and Par4 -/- Mice.
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Experiments in mouse models of thrombosis using arterial injury showed that wild-type (WT) mouse arteries clotted while PAR4 deficient mice were protected. In other experiments, platelet-bound fluorescent antibody at the site of artery injury showed that in WT mice, platelets adhered and aggregated at the site of injury, and the thrombus continued to grow into the vessel lumen. In Par4 -/- mice, platelet thrombus formation began normally but the thrombus did not grow away from the site of injury and compromise the vessel lumen (Figure 2).

Further experiments showed that fibrin formation does not require platelet activation by thrombin in this model. Platelet activation in the juxtamural thrombus by thrombin-independent or platelet-independent mechanisms is sufficient. Blocking coagulation or thrombin activity blocks both fibrin formation and platelet activation and impairs hemostasis. Blocking thrombin signaling spares fibrin formation and hemostasis in mouse models.

Figure 3. Will Inhibiting PAR1 be Sufficient to Achieve an Antithrombotic Effect?
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Figure 4.  Thrombin Responses in Par3 -/- Mouse Platelets and PAR1-Inhibited Human Platelets, αIIb/β3 Activation.
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Figure 5.  Like Par4 -/- mice, Par3 -/- mice are Protected Against Carotid Thrombosis in Response to 4% FeCl3 Injury.
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Par3 -/- mice were used to determine if PAR1 inhibition is sufficient to achieve an antithrombotic effect. PAR1-inhibited human platelets and Par3 -/- mouse platelets both rely on PAR4 for platelet activation by thrombin (Figure 3). Both show a right shift in the concentration responses to thrombin as well as delayed responses. PAR3 deficiency in mouse platelets and PAR1-inhibition in human platelets using SCH530348 both caused a right shift in the concentration response to thrombin (Figure 4). Adding apyrase (APY) to eliminate ADP signaling decreased all responses but the Par3 -/- platelet response was still right-shifted compared to WT and PAR1-inhibited human platelets were still right-shifted compared to untreated platelets. Thus, thrombin responses in Par3 -/- mouse platelets and PAR1-inhibited human platelets are similar. PAR1-inhibition may have an effect even when ADP signaling is already blocked.

Both Par4 -/- and Par3 -/- mice are almost fully protected against carotid thrombosis after arterial injury (Figure 5). P2ry12 mutants are shown for comparison. P2ry12 is the target of clopidogrel, a standard dose of which blocks half of P2ry12 receptors on human platelets. Studies in non-human primates also support the concept that PAR1 inhibition might have an antithrombotic effect.

In a phase 2 study in patients undergoing elective coronary stenting, the PAR1 inhibitor SCH530348 was used as an adjunct to standard antithrombotic therapy or placebo. There was no increase in TIMI bleeding with SCH530348. Although not powered to detect efficacy, the rate of small MIs was 7.3% with placebo versus 3.5% with the highest SCH530348 dose. Two large phase 3 trials of SCH530348 for treatment and prevention of atherothrombotic events are currently ongoing (TRA-CER and TRA2P-TIMI50).

PARs mediate activation of platelets by thrombin. Ablation of PAR function and, hence, thrombin signaling in mouse platelets did not prevent formation of small intra/juxtamural thrombi, did not decrease fibrin formation, and did not cause spontaneous bleeding. Ablation of PAR function in mouse platelets did reduce formation of large platelet thrombi in several thrombosis models. Even partial inhibition of thrombin signaling had substantial effects in low injury models.

These and other studies provide a rationale for exploring inhibition of PAR1 to reduce arterial thrombosis. PAR1 antagonists are currently being tested as an adjunct to standard antithrombotic therapy. The question of whether PAR1 inhibition will provide incremental protection with relative safety as the animal models predict remains open.

 
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