Effect of clopidogrel administration to healthy volunteers on platelet phosphorylation events triggered by ADP

Abstract

Summary. The action of clopidogrel on platelet receptors was analysed using platelets obtained from 11 healthy volunteers given 75 mg of clopidogrel daily for 8 d. Samples of blood were taken before treatment and after 8 d of medication. Determination of 2-methylthioadenosine diphosphate trisodium (2MesADP)-induced platelet aggregation, serine/threonine and tyrosine phosphorylations were performed in the absence or presence of the P2Y1-receptor-specific antagonist: adenosine 3¢-phosphate 5¢-phosphate (A3P5P) or the strong inhibitor of GPIIb/IIIa activation: SR121566. Major conclusions: 1) Serine and threonine phosphorylations of the myosin light chain (P20) and pleckstrin (P47) do not behave similarly, although they are both recognized as the result of phospholipase C pathway stimulation triggered by the P2Y1 receptor. P47 is strongly affected by the A3P5P, and this appears to be highly dependent on P2Y12. However, P20 phosphorylation occurs in the presence of A3P5P, suggesting that the P2Y12 receptor signal contributes to P20 phosphorylation mediated by a calcium-independent pathway. The results suggest that P2Y1 and P2Y12 receptors interact to modulate the phosphorylation of P20 and P47.2) The inside-out signalling dependent on both P2Y12 and P2Y1 is necessary for GPIIb/IIIa activation. 3) Clopidogrel and SR121566 inhibited the increase in tyrosine phosphorylation induced by 2MesADP and concomitantly inhibited platelet aggregation, indicating that most of the phosphorylations are GPIIb/IIIa dependent. However, neither clopidogrel nor SR121566 inhibited the first wave of 80 kDa substrate (cortactin) which is involved in the reorganization of the cytoskeleton necessary for shape change and which appeared to be essentially P2Y1 dependent.

Deciphering of ADP‐induced, phosphotyrosine‐dependent signaling networks in human platelets by Src‐homology 2 region (SH2)‐profiling

“…ADP‐dependent induction of tyrosine phosphorylation was strongly impaired by MRS 2179 treatment while cangrelor had no significant effect. In contrast to our data, a major role of P2Y12 and integrin outside‐in signaling on tyrosine phosphorylation has been proposed previously . Yet in the same place, SRC‐mediated tyrosine phosphorylation of cortactin has been reported being affected by a P2Y1 antagonist.…”

“…Over 50% (64 of 112) of protein bands showed an increase of tyrosine phosphorylation after ADP stimulation, a decrease in phosphorylation was observed for six protein bands and the phosphorylation state of approximately 38% of protein bands remained unaffected indicating that changes in the tyrosine phosphoproteome induced by ADP are highly selective and directed toward specific classes of signaling and effector proteins. In accordance to our results, rapid induction of tyrosine phosphorylation has previously been described after treatment of platelets by ADP or 2-MeSADP, a strong P2 purinergic receptor agonist [37,38]. Using antiphosphotyrosine antibodies or radioactive ( 32 P)-labeling of platelets, several phosphoproteins in the range of 20-130 kDa were detected in these studies.…”

P2Y12 ADP receptor-dependent tyrosine phosphorylation of proteins of 27 and 31 kDa in thrombin-stimulated human platelets

“…Platelets from apatientwithaninherited bleeding disorder associated withadefectin the P2Y12receptor-encoding gene (8,9) showanimpaired proteintyrosinephosphorylation profile in responsetop lateletagonists suchast hrombin and ADP (10). Similarfindingsw ereo btained withADP-triggered platelets from rats and humansafterclopidogrel administration (11,12).…”

“…Inthepresentstudy withwashedh umanp latelets wef ocusedo nADP receptordependentprotein tyrosine phosphorylation in responseto thrombin. Severalchangesin the profile of thrombin-induced protein tyrosinep hosphorylation in humanp latelets havebeen associ-atedwithP2Y12r eceptors ignaling ( 10,12). Along thisline, studieswithplatelets from apatientwithaseveredecreaseinthe numberof ADP-containing densegranules(knownas δ -storagepool disease) revealedtheirnecessity fort hrombin-and collagen-induced aggregation and proteintyrosinephosphorylation (23).…”

“…Insummary,thesereportss howt hatP2Y12r eceptorsignaling affects tyrosinephosphorylation of proteinsof 55,64,80(identifiedascortactin [13]),100,105,125/130 and 140kDa.With platelets from patients withGlanzmann's thrombasthenia (13)as wellasw ithwithp latelets from clopidogrel-treated volunteers (12),ithasbeen demonstrated thattyrosine phosphorylation of p64,cortactin,and p100/105isduetointegrin α IIb β 3 activation and fibrinogen-mediatedoutside-in signaling. Thus,atleast two categoriesof mechanismsr egulating protein tyrosine phosphorylation mediated byP2Y12receptorsignalingmaybedifferentiated:mechanismst hatared irectlyassociated withp athways downstreamo fthe P2Y12r eceptor,a nd thosewhichare subsequentlyaffected,suchasintegrin α IIb β 3 outside-in signaling.…”