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αIIbβ3, the major platelet integrin, plays a central role in hemostasis and thrombosis. Upon platelet activation, conformation of αIIbβ3changes and allows fibrinogen binding and, subsequently, platelet aggregation. It was previously shown that a lipid-modified platelet permeable peptide, which corresponds to the intracellular acidic membrane distal sequence1000LEEDDEEGE1008of αIIb(pal-K-LEEDDEEGE or pal-K-1000-1008), inhibits thrombin-induced human platelet aggregation, by inhibiting talin association with the integrin. YMESRADR, a peptide corresponding to the extracellular sequence 313–320 of αIIb, is also a potent platelet aggregation inhibitor by mimicking the effect of a clasp between the head domains of αIIband β3. The aim of the present study was to investigate the synergistic effect of the intra- and extracellular- peptide inhibitors on platelet aggregation, as well as on the phosphorylation of two signaling proteins, focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK). Platelet preincubation with Pal-K-LEEDDEGE followed by YMESRADR showed a synergistic inhibitory activity on platelet aggregation. Platelet incubation with threshold inhibitory concentrations of both peptides provoked almost the total inhibition of aggregation, PAC-1 binding, and fibrinogen binding, but not P-selectin exposure on activated platelets’ surface. Like RGDS peptide, this mixture inhibits FAK phosphorylation whose phosphorylation is well known to be consecutive to the aggregation (postoccupancy events). However, in contrast to RGDS peptide that enhances ERK phosphorylation and activation, the mixture of threshold inhibitory concentrations of Pal-K-LEEDDEEGE and YMESRADR inhibits ERK phosphorylation. We suggest that the use of the intracellular in combination with the extracellular peptide inhibitor, acting with a non-RGD-like mechanism, may provide an alternative way to antagonize integrin αIIbβ3activation. [ABSTRACT FROM PUBLISHER]

The αIIb cytoplasmic domain of platelet integrin αIIbβ3 contains an unorganized acidic membrane-distal 1000LEEDDEEGE1008 region. We have shown that a platelet permeable peptide corresponding to the above region the palmitoyl-K-LEEDDEEGE (pal-K-1000-1008) inhibits platelet aggregation induced by thrombin or by pal-K-989-995, a palmitoylated peptide corresponding to the membrane-proximal αIIb cytoplasmic domain 989KVGFFKR995. We now tested the anti-aggregatory activity of (i) a lipid-modified scrambled acidic peptide (pal-K-GDDEELEEE), (ii) two smaller peptides derived from the acidic amino sequence: palmitoyl-K-1000LEEDDE1005 (pal-K-1000-1005) and palmitoyl-K-1005EEGE1008 (pal-K-1005-1008) and (iii) lipid-modified palmitoyl-acidic peptides with alanine (Ala) substitution at residues 1001, 1003, 1004 and 1005 and one peptide with a double Ala substitution at residues 1001 and 1004 of the 1000-1008 sequence. All the peptides tested showed an inhibitory activity, however, the palmitoylated peptide with the natural and the whole acidic sequence, being the most active. Our results suggest that the whole acidic sequence, rather than some specific amino acids, contributes to the aggregation inhibitory activity. The inhibitory peptide, pal-K-1000-1008, inhibited the association of talin with αIIbβ3 in thrombin-activated platelets, as demonstrated by co-immunoprecipitation experiments, while the scrambled peptide was inefficient. We suggest that, by interacting with αIIb cytoplasmic domain, pal-K-1000-1008 has an anti-aggregatory inhibitory activity due to a specific inhibition of talin binding to αIIbβ3. [ABSTRACT FROM AUTHOR]

Platelet integrin αIIbβ3 contains an acidic membrane distal motif, 1000LEEDDEEGE1008, in the cytoplasmic domain of the αIIb subunit. We showed that a lipid-modified peptide corresponding to the above region, palmitoyl-K-LEEDDEEGE (pal-K-1000-1008), is platelet permeable and has inhibited platelet aggregation induced by 0.4 U/ml of thrombin (IC50 = 164 µM). Moreover the peptide inhibited both Fibrinogen and PAC-1, binding to activated platelets. The non palmitoylated analog was inactive. A modified, scrambled acidic peptide (palmitoyl-K-GDDEELEEE), showed significant lower inhibitory activity than pal-K-1000-1008. A palmitoylated peptide corresponding to the membrane proximal cytoplasmic domain of αIIb, 989KGVFFKR995 (pal-989-995), is known to specifically induce platelet aggregation. Pal-K-1000-1008 was an inhibitor of human washed platelet aggregation induced by pal-K-989-995 (IC50 = 15 µM). Moreover, pal-K-1000-1008 inhibited phosphorylation of ERK and FAK, two protein kinases involved in platelet activation and aggregation. Our results favour the assumption that the interaction of the membrane proximal sequence 989KGVFFKR995 of the cytoplasmic domain of αIIb with the acidic terminal 1000LEEDDEEGE1008 motif may be an important structural factor in platelet signaling, leading to platelet activation and aggregation. [ABSTRACT FROM AUTHOR]

Objective: Platelet activation and aggregation is a dominant feature in the pathophysiology of unstable angina. The final step of platelet aggregation is mediated through the platelet integrin glycoprotein IIb/IIIa (GP IIb/IIIa), while abciximab (ReoPro) is one of the most potent inhibitors of this receptor. Platelet-activating factor (PAF) is a potent platelet agonist which is degraded and inactivated by PAF-acetylhydrolase (PAF-AH). The plasma form of PAF-AH is associated with lipoproteins. We studied the platelet response to the aggregatory effect of PAF, ex vivo, in relation to the plasma PAF-AH activity in 32 patients with unstable angina, as well as the effect of abciximab therapy on the above parameters. Methods: Thirty two patients with unstable angina and 25 sex- and age-matched healthy controls participated in the study. On the day of admission (day 1) 17 patients received a bolus of abciximab (0.25 mg/kg) followed by a 12-h infusion (10 μg/min). Platelet aggregation to both PAF and ADP, in platelet rich plasma, was successively studied in both patients receiving abciximab or remaining untreated. The plasma and HDL-associated PAF-AH activity was also determined at the same times. Results: In the untreated patients, the PAF EC50 values were significantly lower on the day of admission, whereas the maximal percentage of aggregation was significantly higher compared to controls (p<0.01 for both comparisons). Similar behaviour of the platelets was observed in the aggregatory effect of ADP. This aggregatory response was not significantly altered 4 days, 7 days or 1 month afterwards. In the 17 patients who received abciximab, platelet aggregation to both PAF and ADP was inhibited by 90±5 and 96±3%, respectively, 1 h after bolus. At 2 and 3 days after treatment, platelet aggregation to both agonists was significantly recovered being similar to controls. However, it was fully restored 6 days after bolus, still being significantly higher compared to controls (p<0.01 for PAF and p<0.003 for ADP). The total plasma PAF-AH activity in both patient groups was not different from that of controls, whereas the HDL-associated PAF-AH activity was significantly lower. The total plasma or HDL-associated enzyme activity was not altered at any time interval studied, and it was not influenced by abciximab. Conclusions: The increased aggregatory response of platelets to PAF and the low plasma levels of HDL-cholesterol and HDL-associated PAF-AH activity in patients with unstable angina may contribute to the severe atherosclerosis and to acute thrombosis found in these patients. Abciximab therapy may protect platelets from PAF action in vivo the first days after drug administration, but it fails to permanently restore the enhanced aggregatory response observed. [ABSTRACT FROM PUBLISHER]

The αIIb cytoplasmic domain of platelet integrin αIIbβ3 contains an unorganized acidic membrane-distal 1000LEEDDEEGE1008 region. We have shown that a platelet permeable peptide corresponding to the above region the palmitoyl-K-LEEDDEEGE (pal-K-1000-1008) inhibits platelet aggregation induced by thrombin or by pal-K-989-995, a palmitoylated peptide corresponding to the membrane-proximal αIIb cytoplasmic domain 989KVGFFKR995. We now tested the anti-aggregatory activity of (i) a lipid-modified scrambled acidic peptide (pal-K-GDDEELEEE), (ii) two smaller peptides derived from the acidic amino sequence: palmitoyl-K-1000LEEDDE1005 (pal-K-1000-1005) and palmitoyl-K-1005EEGE1008 (pal-K-1005-1008) and (iii) lipid-modified palmitoyl-acidic peptides with alanine (Ala) substitution at residues 1001, 1003, 1004 and 1005 and one peptide with a double Ala substitution at residues 1001 and 1004 of the 1000-1008 sequence. All the peptides tested showed an inhibitory activity, however, the palmitoylated peptide with the natural and the whole acidic sequence, being the most active. Our results suggest that the whole acidic sequence, rather than some specific amino acids, contributes to the aggregation inhibitory activity. The inhibitory peptide, pal-K-1000-1008, inhibited the association of talin with αIIbβ3 in thrombin-activated platelets, as demonstrated by co-immunoprecipitation experiments, while the scrambled peptide was inefficient. We suggest that, by interacting with αIIb cytoplasmic domain, pal-K-1000-1008 has an anti-aggregatory inhibitory activity due to a specific inhibition of talin binding to αIIbβ3. [ABSTRACT FROM AUTHOR]

The αIIb cytoplasmic domain of platelet integrin αIIbβ3 contains an unorganized acidic membrane-distal (1000)LEEDDEEGE(1008) region. We have shown that a platelet permeable peptide corresponding to the above region the palmitoyl-K-LEEDDEEGE (pal-K-1000-1008) inhibits platelet aggregation induced by thrombin or by pal-K-989-995, a palmitoylated peptide corresponding to the membrane-proximal αIIb cytoplasmic domain (989)KVGFFKR(995). We now tested the anti-aggregatory activity of (i) a lipid-modified scrambled acidic peptide (pal-K-GDDEELEEE), (ii) two smaller peptides derived from the acidic amino sequence: palmitoyl-K-(1000)LEEDDE(1005) (pal-K-1000-1005) and palmitoyl-K-(1005)EEGE(1008) (pal-K-1005-1008) and (iii) lipid-modified palmitoyl-acidic peptides with alanine (Ala) substitution at residues 1001, 1003, 1004 and 1005 and one peptide with a double Ala substitution at residues 1001 and 1004 of the 1000-1008 sequence. All the peptides tested showed an inhibitory activity, however, the palmitoylated peptide with the natural and the whole acidic sequence, being the most active. Our results suggest that the whole acidic sequence, rather than some specific amino acids, contributes to the aggregation inhibitory activity. The inhibitory peptide, pal-K-1000-1008, inhibited the association of talin with αIIbβ3 in thrombin-activated platelets, as demonstrated by co-immunoprecipitation experiments, while the scrambled peptide was inefficient. We suggest that, by interacting with αIIb cytoplasmic domain, pal-K-1000-1008 has an anti-aggregatory inhibitory activity due to a specific inhibition of talin binding to αIIbβ3.

Platelet integrin alpha(IIb)beta(3) contains an acidic membrane distal motif, 1000LEEDDEEGE1008, in the cytoplasmic domain of the alpha(IIb) subunit. We showed that a lipid-modified peptide corresponding to the above region, palmitoyl-K-LEEDDEEGE (pal-K-1000-1008), is platelet permeable and has inhibited platelet aggregation induced by 0.4 U/ml of thrombin (IC50 = 164 microM). Moreover the peptide inhibited both Fibrinogen and PAC-1, binding to activated platelets. The non palmitoylated analog was inactive. A modified, scrambled acidic peptide (palmitoyl-K-GDDEELEEE), showed significant lower inhibitory activity than pal-K-1000-1008. A palmitoylated peptide corresponding to the membrane proximal cytoplasmic domain of alpha(IIb), 989KGVFFKR995 (pal-989-995), is known to specifically induce platelet aggregation. Pal-K-1000-1008 was an inhibitor of human washed platelet aggregation induced by pal-K-989-995 (IC50 = 15 microM). Moreover, pal-K-1000-1008 inhibited phosphorylation of ERK and FAK, two protein kinases involved in platelet activation and aggregation. Our results favour the assumption that the interaction of the membrane proximal sequence 989KGVFFKR995 of the cytoplasmic domain of alpha(IIb) with the acidic terminal 1000LEEDDEEGE1008 motif may be an important structural factor in platelet signaling, leading to platelet activation and aggregation.