The Direct Thrombin Inhibitors Dabigatran and Lepirudin Inhibit GPIbα-Mediated Platelet Aggregation

Abstract

The direct thrombin inhibitor (DTI) dabigatran is a non-vitamin K antagonist oral anticoagulant for the prevention of stroke and systemic embolism in patients with non-valvular atrial fibrillation. In addition to its anti-thrombotic efficacy, dabigatran has been suggested to exert some pro-thrombotic effect due to fostering the ligation of thrombin to its high affinity platelet receptor glycoprotein (GP) Ibα in patients with atrial fibrillation. On the other hand, we provided evidence that a member of another class of DTIs, lepirudin, stimulates the inhibitory cyclic guanosine monophosphate (cGMP)/soluble guanylate cyclase pathway in human platelets. Here, we investigated the effect of lepirudin and dabigatran spiked to platelets from healthy volunteers on GPIbα-mediated platelet aggregation and agglutination. Ristocetin/von Willebrand factor (vWF)-induced aggregation of platelets in the presence or absence of plasma was significantly inhibited by lepirudin, dabigatran and D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone (PPACK). However, ristocetin/vWF-mediated platelet agglutination and binding of vWF to platelets were not affected by the DTIs. The anti-aggregatory effect was confirmed by using the GPIbα-specific agonist echicetin beads for human and murine platelets. DTIs diminished echicetin beads-induced Syk Y352 phosphorylation (used here as readout for an early signal occurring during echicetin-induced platelet aggregation), but did not inhibit adenosine diphosphate- or thromboxane A2-induced platelet aggregation. Thrombin was not generated in response to ristocetin/vWF or echicetin beads and therefore did not explain the inhibitory effect of the DTIs. Therapeutic concentration of lepirudin and dabigatran did not affect significantly platelet vasodilator-stimulated phosphoprotein S239 phosphorylation or cGMP and cyclic adenosine monophosphate levels. These data suggest that the DTIs, lepirudin and dabigatran, impair platelet activation measured during platelet aggregation induced by ristocetin/vWF or echicetin beads.

cAMP- and cGMP-elevating agents inhibit GPIbα-mediated aggregation but not GPIbα-stimulated Syk activation in human platelets

“…The platelet pellet was resuspended with Hepes buffer (150 mM NaCl, 5 mM KCl, 1 mM MgCl 2 , 10 mM Glucose, 10 mM Hepes) pH 7.4. Agglutination followed by fibrinogen-dependent aggregation of washed human platelets was induced by adding human vWF (2.5 μg/ml final concentration) plus ristocetin (0.5 mg/ml final concentration) as previously described [41]. Residual platelet aggregation was calculated by subtraction of overall maximum light transmission in the absence of tirofiban minus maximum light transmission in the presence of tirofiban.…”

“…Earlier studies showed that echicetin molecules cross-linked by plasma IgMκ caused platelet agglutination and weak aggregation whereas echicetin monomers, when used as specific GPIbα ligands, competed with vWF and thrombin for binding to GPIbα and blocked ristocetin/vWF mediated platelet agglutination [40]. Using a transgenic mouse model, we recently reported that the extracellular GPIbα-domain of murine platelets is essential for echicetin-bead-induced platelet aggregation [41]. In this model the extracellular domain of GPIbα is replaced by the human interleukin 4-receptor (IL4/GPIbα transgenic mice).…”

Feedback Regulation of Syk by Protein Kinase C in Human Platelets

“…Interestingly, ADP, which activates platelet Ca 2+ /calmodulin-dependent protein kinases such as PKC, but not iloprost, stimulated Syk S297 phosphorylation. Very recently, we established methods for the selective quantitative assessment of GPVI-and GPIbα-mediated activation and function of human platelet Syk [27,28]. We observed that cAMP-and cyclic guanosine monophosphate (cGMP)-elevating platelet inhibitors strongly inhibited GPIbα-/GPVI-mediated platelet activation but enhanced the initial Syk activation [28].…”

Noncanonical Effects of Oral Thrombin and Factor Xa Inhibitors in Platelet Activation and Arterial Thrombosis

“…30 In contrast, a recent analysis conducted by Trabold et al did not observe increased vWF/ristocetin-induced platelet aggregation. 31 To discuss potential reasons of the discrepant results, it is crucial to appreciate the different experimental setups used in either study. Trabold et al investigated platelets and platelet-rich plasma from healthy volunteers spiked with dabigatran and other divalent thrombin inhibitors, while our group used whole blood from patients under chronic therapy.…”