Antiplatelet effect of clopidogrel in patients with aspirin therapy undergoing percutaneous coronary interventions--limited inhibition of the P2Y12 receptor.

INTRODUCTION: Large individual variability in clopidogrel responses has been reported. However, mechanisms of the non-responsiveness are unclear. Our aim was to study the extent of platelet inhibition at the receptor level by in vitro receptor antagonists of P2Y(12) (AR-C69931MX, cangrelor) and P2Y(1) (adenosine 3',5'diphosphate) in aspirin treated patients with coronary artery disease (CAD) prior to and after in vivo clopidogrel. MATERIALS AND METHODS: 51 aspirin-treated (100 mg/day) patients participated. Blood was collected before and after administration of clopidogrel at 300 mg loading dose on day one, followed by 75 mg/d for four days. Aggregation in platelet-rich plasma was assessed. RESULTS: In 20% of patients clopidogrel failed to inhibit platelet responses to ADP. These non-responders had also decreased sensitivity to an in vitro P2Y(12)-receptor antagonist compared with the responders (mean inhibition of aggregation 25 vs. 32%, difference of means 7% (95% CI 2-12%), P<0.02). Moreover, the P2Y(12)-receptor inhibition by in vivo clopidogrel correlated with the inhibition by in vitro ARMX measured prior to administration of clopidogrel. Neither P2Y(1)-receptor activity, thrombin generation while on aspirin nor basal platelet activity associated with clopidogrel responses. CONCLUSIONS: Concomitant aspirin and clopidogrel treatment failed to suppress platelet activity in 20% of patients. Non-responders to clopidogrel had decreased responses also to another ADP receptor antagonist, which suggests that the impaired response occurs at the level of P2Y(12)-receptor.

Platelet microparticle suppressing antibody against GP Ibalpha acts independently of the filamin cleavage and increases protein tyrosine phosphorylation.

Our aim was to investigate the role of filamin cleavage and protein tyrosine phosphorylation in shear-stress-induced platelet microparticle formation and of its suppression by the monoclonal antibody (mAb) Ib-23 directed against GPIbalpha. PPACK-anticoagulated or EDTA-anticoagulated platelet-rich plasma or washed platelets were exposed to high shear stress (5000 s-1 for 5 min) in the presence of antagonists of GP Ibalpha (mAb Ib-23), of GP IIb/IIIa (abciximab) and their combination. We assessed the generation of microparticle by flow cytometry, the filamin cleavage and the protein tyrosine phosphorylation by western blotting. Microparticle formation decreased by more than 50% with mAb Ib-23 alone (P < 0.01, n = 23) but filamin cleavage was not inhibited. In contrast, abciximab did not change microparticle numbers nor filamin cleavage under the same conditions. However, when combined, the inhibitors against GP Ibalpha and GP IIb/IIIa decreased microparticle by 70% and the filamin cleavage by 20% (P < 0.05). Focal adhesion kinase and p72Syk phosphorylation was enhanced by mAb Ib-23, whereas treatment with abciximab reduced it. mAb Ib-23 inhibits platelet microparticle induced by high shear stress. The mechanism seems not to depend on filamin cleavage: abciximab allowed for full microparticle formation at similar levels of cleaved filamin, whereas the combined inhibitors reduced it. The effect of mAb Ib-23 on protein tyrosine phosphorylation supports previous data, which correlates microparticle formation with the extent of protein tyrosine dephosphorylation and mirrors the dephosphorylation by protein tyrosine phosphatase (PTP-1B) in platelets of calpain-/- knockout mice.

Improved identification of endothelial erosion by simultaneous detection of endothelial cells (CD31/CD34) and platelets (CD42b).

Loss of endothelial cells (ECs) with ensuing exposure of thrombogenic subendothelial surface is a common cause of thromboembolic complications in atherosclerotic arteries. Thus, endothelial denudation has emerged as a major contributor to the pathogenesis of atherosclerosis and its complications. Despite ongoing efforts in elucidating the pathogenesis of endothelial erosions in human atherosclerotic arteries, the mechanisms of erosion have remained enigmatic, partly due to lack of well-established methods for its identification. Here the authors point out plausible pitfalls in the current methodology and provide an improved immunohistochemical method for identifying endothelial erosion; i.e., immunofluorescence double staining with antibodies against CD42b and CD31/CD34. This method enables reliable detection of ECs and platelets in the same staining by allowing detection of "pseudoendothelium" caused by CD31 staining of a thin platelet layer covering sites of endothelial erosion. As erosion with a luminal platelet thrombus is likely to represent an in vivo erosion, and erosion without platelets an ex vivo artefact, the method makes it possible to exclude artefactual erosions resulting from sample processing. The novel immunostaining protocol presented here allows more reliable detection of endothelial erosions and so may facilitate studies on the mechanisms involved in the pathogenesis of plaque erosion and acute coronary syndromes.

Pathological shear stress directly regulates platelet alphaIIbbeta3 signaling.

Integrin mechanotransduction is a ubiquitous biological process. Mechanical forces are transduced transmembranously by an integrin's ligand-bound extracellular domain through its beta-subunit's cytoplasmic domain connected to the cytoskeleton. This often culminates in the activation of tyrosine kinases directing cell responses. The delicate balance between hemostasis and thrombosis requires exquisitely fine-tuned integrin function, and balance is maintained in vivo despite that the major platelet integrin alpha(IIb)beta(3) is continuously subjected to frictional or shearing forces generated by laminar blood flow. To test the hypothesis that platelet function is regulated by the direct effects of mechanical forces on alpha(IIb)beta(3), we examined alpha(IIb)beta(3)/cytoskeletal interactions in human platelets exposed to shear stress in a cone-plate viscometer. We observed that alpha-actinin, myosin heavy chain, and Syk coimmunoprecipitate with alpha(IIb)beta(3) in resting platelets and that 120 dyn/cm(2) shear stress leads to their disassociation from alpha(IIb)beta(3). Shear-induced disassociation of alpha-actinin and myosin heavy chain from the beta(3) tail is unaffected by blocking von Willebrand factor (VWF) binding to glycoprotein (Gp) Ib-IX-V but abolished by blocking VWF binding to alpha(IIb)beta(3). Syk's disassociation from beta(3) is inhibited when VWF binding to either GpIb-IX-V or alpha(IIb)beta(3) is blocked. Shear stress-induced phosphorylation of SLP-76 and its association with tyrosine-phosphorylated adhesion and degranulation-promoting adapter protein are inhibited by blocking ligand binding to alpha(IIb)beta(3) but not by blocking ligand binding to GpIb-IX-V. Chinese hamster ovary cells expressing alpha(IIb)beta(3) with beta(3) truncated of its cytoskeletal binding domains demonstrate diminished shear-dependent adhesion and cohesion. These results support the hypothesis that shear stress directly modulates alpha(IIb)beta(3) function and suggest that shear-induced alpha(IIb)beta(3)-mediated signaling contributes to the regulation of platelet aggregation by directing the release of constraining cytoskeletal elements from the beta(3)-tail.

Polymorphisms of COX-1 and GPVI associate with the antiplatelet effect of aspirin in coronary artery disease patients.

The antiplatelet effect of aspirin varies individually. This study evaluated whether the antiplatelet effect of aspirin associates with polymorphisms in the genes coding for cyclo-oxygenase-1 (COX-1) and several platelet glycoprotein (GP) receptors in patients with stable coronary artery disease (CAD). Blood samples were collected from 101 aspirin-treated (mean 100 mg/d) patients. Compliance to treatment was assessed by plasma salicylate measurement. Platelet functions were assessed by two methods: 1) Response to arachidonic acid (AA, 1.5 mmol/L in aggregometry, and 2) PFA-100, evaluating platelet activation under high shear stress in the presence of collagen and epinephrine (CEPI). Aspirin non-response was defined as: 1) slope steeper than 12%/min in AA-aggregations, and 2) by closure time shorter than 170 s in PFA-100. The methods used detected different individuals as being aspirin non-responders. Five and 21 patients, respectively, were non-responders according to AA-induced aggregation and PFA-100. Increased plasma thromboxane B2 levels correlated with poor aspirin-response measured with both AA-induced aggregations and PFA-100 (P = 0.02 and P = 0.003, respectively). Of the non-responders detected by AA, 3 of 5 (60%) carried the rare G allele for the -A842G polymorphism of COX-1 in contrast to 16 of 96 (17%) responders (P = 0.016). Diabetes was associated with poor response. Aspirin non-response detected by PFA-100 associated with C13254T polymorphism of GP VI and female gender (P = 0.012 and P = 0.019, respectively). Although two patients were possibly non-compliant, this did not effect present conclusions. Evaluation of aspirin efficacy by AA-induced aggregation and PFA-100 detected different individuals, with different genotypic profiles, as being aspirin non-responders.

Role of the Pyk2-MAP kinase-cPLA2 signaling pathway in shear-dependent platelet aggregation.

Mechanisms of shear-induced platelet aggregation are not established. Data that ristocetin-induced von Willebrand factor (VWF) binding to glycoprotein (Gp) Ibalpha activates proline-rich tyrosine kinase 2 (Pyk2) and extracellular-regulated kinase (ERK) has led to speculation that these events are coupled and that a MAP kinase may activate cytosolic phospholipase A2 (cPLA2)-mediated arachidonic acid (AA) release. To test this hypothesis and clarify the role of AA metabolism in shear-induced VWF-dependent platelet aggregation, we examined Pyk2, ERK1/2, and p38 phosphorylation, and arachidonic acid release and metabolism in platelets subjected to pathological shear stress in vitro. We observe tyrosine phosphorylation of Pyk2, p38, and ERK1/2 but no measurable increase in free AA, 12-hydroxyeicosatetraenoic acid, or thromboxane A2. Inhibitors of ERK, p38, or cyclooxygenase activation fail to affect shear-induced platelet aggregation. When washed platelets are aspirin-pretreated, arachidonic acid release becomes measurable and aggregation at 60 and 120 s is attenuated. These data indicate that shear-induced VWF binding to platelet GpIb-IX-V activates Pyk2, ERK1/2, p38, and cPLA2, but that the magnitude of these responses is below the threshold needed to enhance shear-induced VWF-dependent platelet aggregation in the presence of plasma. These results provide a mechanistic basis for the long-standing observation that shear-dependent platelet aggregation is unaffected by the antiplatelet drug aspirin.

Filamin A binding to the cytoplasmic tail of glycoprotein Ibalpha regulates von Willebrand factor-induced platelet activation.

We examined the hypothesis that filamin A binding to the cytoplasmic tail of platelet glycoprotein Ibalpha (GpIbalpha) is regulated by pathologic shear stress and modulates von Willebrand factor (VWF)-induced platelet activation. To begin, we examined filamin binding to GpIbalpha in Chinese hamster ovary cells coexpressing mutant human GpIb-IX and wild-type human filamin A. We observed that many different deletions and truncations N-terminal to GpIbalpha's cytoplasmic domain residue 594 disrupted filamin A binding, but that binding was unaffected by 14 different point mutations in hydrophilic residues between amino acids 557 and 593. To try to narrow GpIbalpha's filamin A-binding domain, we next measured the effect of several cytoplasmic domain peptides on human filamin A binding to a GST-GpIbalpha cytoplasmic domain fusion protein. One peptide (residues 557-575; designated "A4 peptide") inhibited filamin A binding to the GST-GpIbalpha cytoplasmic domain fusion protein and competed with GpIbalpha for binding to filamin A. When the A4 peptide was delivered to intact human platelets using a carrier peptide, we observed the dose-dependent inhibition of VWF-induced platelet aggregation in response to both ristocetin and shear stress. The effect of the A4 peptide on shear-induced platelet aggregation was accompanied by the attenuation of shear-induced filamin A binding to GpIbalpha and diminished shear-dependent protein tyrosine phosphorylation. These results suggest that shear-dependent VWF-induced platelet activation affects filamin A binding to GpIb-IX-V, and that filamin A binding to the cytoplasmic tail of GpIbalpha regulates proaggregatory tyrosine kinase signaling.

Purinergic P2Y12 receptor blockade inhibits shear-induced platelet phosphatidylinositol 3-kinase activation.

Pathologically elevated shear stress triggers aspirin-insensitive platelet thrombosis. Signaling mechanisms involved in shear-induced platelet thrombosis are not well understood. To investigate these, we examined the hypothesis that functionally important platelet phosphatidylinositol 3-kinase (PI3-K) activity is stimulated by an in vitro shear stress of 120 dynes/cm(2) (shear rate of 6,000 sec(-1)). Phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) production was examined in washed human platelets subjected to pathological shear stress in a cone-plate viscometer. PIP(3) production peaks 30 s after shear begins and is initiated by von Willebrand factor (VWF) binding to the glycoprotein (Gp) Ib-IX-V complex. Inhibiting PI3-K with wortmannin or 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002) results in the inhibition of shear-induced platelet aggregation. In resting platelets, class IA PI3-K associates with the tyrosine kinase Syk. Within 30 s of beginning shear, PI3-K-associated Syk becomes tyrosine phosphorylated. Inhibiting Syk activation with piceatannol results in the inhibition of PIP(3) production and aggregation. Selective blockade of the P2Y(12) receptor results in the inhibition of Syk phosphorylation, PIP(3) production, and aggregation. These results indicate that shear-induced VWF binding to platelet GpIb-IX-V stimulates functionally important PI3-K activity. PI3-K activation is signaled by rapid feedback amplification that involves P2Y(12) receptor-mediated activation of Syk.

Pathological shear stress stimulates the tyrosine phosphorylation of alpha-actinin associated with the glycoprotein Ib-IX complex.

Shear-induced platelet responses are triggered by VWF binding to the platelet GpIb-IX complex, and there is evidence that this ligand-receptor coupling stimulates transmembranous signaling through the cytoplasmic tail of glycoprotein (Gp) Ib alpha. To investigate the mechanism by which signaling is effected, new molecular interactions involving GpIb-IX that develop in response to pathological shearing stress were examined in intact human platelets. Exposure to shear, but not alpha-thrombin, results in the co-immunoprecipitation of the actin cross-linking protein alpha-actinin with the GpIb-IX complex. Blockers of VWF binding to GpIb alpha or actin polymerization inhibit the association of alpha-actinin with the GpIb-IX complex, but the association of alpha-actinin with the GpIb-IX complex is not affected by inhibiting VWF binding to platelet integrin alpha IIb beta 3 (GpIIb-IIIa). alpha-Actinin becomes tyrosine phosphorylated in response to pathological shear stress, and phosphorylated alpha-actinin associates with GpIb-IX. In resting platelets, class IA heterodimeric phosphatidylinositol 3-kinase (PI 3-K) and protein kinase N (PKN) associate with nonphosphorylated alpha-actinin. Shear stress causes PI 3-K to disassociate from alpha-actinin, while it stimulates PKN binding to alpha-actinin. These results demonstrate that shear-induced VWF binding to GpIb alpha causes enhanced binding of cytoskeletal alpha-actinin to GpIb-IX and suggest that alpha-actinin, perhaps through tyrosine phosphorylation, serves as an adapter for a signaling complex that could regulate VWF-induced platelet aggregation.