The thrombin inhibitors hirudin and Refludan® activate the soluble guanylyl cyclase and the cGMP pathway in washed human platelets

Abstract

A number of direct thrombin inhibitors are successfully used clinically and experimentally as novel antithrombotics and specific anticoagulants. They are also used as anticoagulants in certain blood collection tubes for the analysis of platelet function. A series of platelet function tests have emerged to measure adequate responses to antiplatelet therapy. For comparative and practical reasons, it would be of advantage to use the same anticoagulant in blood collection tubes for different methods, e.g. thrombin inhibitors. However, there are little data on the effects of thrombin inhibitors on platelet signalling pathways that could influence results. We examined the applicability of thrombin inhibitor containing blood for platelet reactivity index (PRI) measurements of the VASP assay and investigated the effects of two thrombin inhibitors (hirudin and lepirudin) on cAMP- and cGMP-mediated signalling pathways in washed human platelets. We show that induction of VASP phosphorylation by PGE1 is markedly reduced in lepirudin containing blood samples. In consequence, PRI levels were highly variable compared to routinely used citrated blood. Surprisingly, in vitro incubation of platelets with thrombin inhibitors increases platelet cGMP levels and induces NOS independent sGC/PKG-mediated VASP phosphorylation. We conclude that thrombin inhibitors activate sGC/PKG-dependent pathways resulting in an increase of VASP phosphorylation which contributes to deviations in PRI measurements. These effects of thrombin inhibitors on sGC- and cGMP-mediated pathways including increased VASP phosphorylation may indicate the presence of an important additional platelet-based mechanism for the reduction of thrombus formation and thromboembolism by thrombin inhibitors.

Platelet Protease Activated Receptor 1 Is Involved in the Hemostatic Effect of 20(S)-Protopanaxadiol by Regulating Calcium Signaling

“…Importantly, PAR-1 is coupled to G q and G i proteins that lead to a reduction in cAMP ( Yang et al., 2002 ), and cGMP concentration is increased by thrombin-induced platelets ( Kobsar et al., 2012 ); therefore, we detected the effect of PPD on the levels of cAMP and cGMP in human/rat washed platelets. As shown in Figures 8A, B , PPD inhibited cAMP production of human platelets in a dose-dependent manner compared with the vehicle group ( p < 0.05 or p < 0.01), which had no effect on cGMP production in human platelets.…”

The Impact of Cold Storage on Adenosine Diphosphate-Mediated Platelet Responsiveness

“…In the context of PRI determination, it is required to closely study the underlying shifts of VASP phosphorylation. 33 34 Thereby, it was evident that stimulation of PGE1-induced VASP phosphorylation in C-APC is decreased, whereas ADP-induced inhibition of VASP phosphorylation remains intact, indicating maintained P2Y12 receptor function.…”

“…Thrombin inhibitors-like hirudin may be alternatives, but there is evidence that thrombin inhibitors also affect inhibitory pathways, thereby enhancing VASP phosphorylation and dampening platelet reactivity. 34 Interestingly, BAPA (benzylsulfonyl-D-Arg-Pro-4-amidinobenzylamide) represents a dual inhibitor of factor Xa and thrombin, was able to maintain platelet aggregation and function in stored blood samples at RT better than citrate, indicating a significant role of the anticoagulant in platelet preservation. 46 47 …”

High Glucose Inhibits the Aspirin-Induced Activation of the Nitric Oxide/cGMP/cGMP-Dependent Protein Kinase Pathway and Does Not Affect the Aspirin-Induced Inhibition of Thromboxane Synthesis in Human Platelets

“…The pellet was washed two times with HEPES-Na buffer (10 mmol/L HEPES Na, 140 mmol/L NaCl, 2.1 mmol/L, MgSO 4 , 5 mmol/L glucose, pH 7.4). WPs are usually used to measure the NO/cGMP/PKG signaling in platelets (33). They present some degree of activation; actually, the concentrations of the platelet activation marker sP-selectin (ELISA kit; Bender MedSystems GmbH, Vienna, Austria) were similar in PRP and in surnatants of WPs (24.2 ± 1.9 and 25.7 ± 1.7 ng/10 8 platelets; n = 7; P = NS), indicating that WPs still release P-selectin after removal of the protein contained in the plasma fraction of PRP.…”