Platelet glycoprotein IIb/IIIa receptor antagonists derived from isoxazolidines

A series of isoxazolidines has been synthesized as mimetics of the RGD sequence and was evaluated as antagonists of the platelet glycoprotein IIb/IIIa receptor. These compounds were shown to be highly potent GPIIb/IIIa antagonists, exhibiting submicromolar potencies.     

Platelet glycoprotein IIb/IIIa receptor blockade in coronary artery disease

Glycoprotein IIb/IIIa inhibitors represent a new promising class of antiplatelet medications. Their use in acute coronary syndromes and for patients undergoing percutaneous coronary intervention has been the subject of a number of large controlled trials using both the intravenous and the oral forms. In this review, we present a systematic overview of these trials.     

Glycoprotein IIb/IIIa receptor antagonists in acute myocardial infarction and primary coronary stenting: Not always effective

Glycoprotein IIb/IIIa antagonists have been shown to be effective in reducing thrombotic complications prior to high-risk coronary interventions. Some studies have reported improved coronary flow after abciximab in slow or no-reflow phenomenon. We report a case in which abciximab did not clear the thrombotic occlusion or restore artery flow. Further studies are needed into the refractory no-flow phenomenon.     

Synthesis and pharmacology of modified amidine isoxazoline glycoprotein IIb/IIIa receptor antagonists

Selective antagonism of the platelet GPIIb/IIIa receptor represents an attractive mechanism for the prevention and treatment of a number of thrombotic disease states. The antiplatelet activity of the oral GPIIb/IIIa receptor antagonists DMP 754 and DMP 802 have been disclosed. In this paper, the synthesis and biological evaluation of a series of potent N-substituted benzamidine isoxazolines are explored. The effect of benzamidine substitution on the duration of antiplatelet efficacy in dog is presented.     

Trigramin: primary structure and its inhibition of von Willebrand factor binding to glycoprotein IIb/IIIa complex on human platelets

Trigramin, a naturally occurring peptide purified from Trimeresurus gramineus (T. stejnegeri formosensis) snake venom, inhibits platelet aggregation and the binding of 125I-fibrinogen to ADP-stimulated platelets (Ki = 2 X 10(-8) M) without affecting the platelet-release reaction. 125I-trigramin binds to ADP-stimulated and to chymotrypsin-treated normal platelets but not to thrombasthenic platelets. 125I-trigramin binding to platelets is blocked by monoclonal antibodies directed against the glycoprotein IIb/IIIa complex and by Arg-Gly-Asp-Ser (RGDS) [Huang et al. (1987) J. Biol. Chem. 262, 161]. We determined the primary structure of trigramin, which is composed of a single polypeptide chain of 72 amino acid residues and six disulfide bridges. The molecular weight of trigramin calculated on the basis of amino acid sequence was 7500, and the average pI was 5.61. An RGD sequence appeared in the carboxy-terminal domain of trigramin. An amino-terminal fragment (7-33) of trigramin showed 39% homology with a region (1555-1581) of von Willebrand factor (vWF). Trigramin also showed 36% identity in a 42 amino acid overlap and 53% identity in a 15 amino acid overlap when compared with two adhesive proteins, collagen alpha 1 (I) and laminin B1, respectively. Trigramin blocked binding of human vWF to the glycoprotein IIb/IIIa complex in thrombin-activated platelets in a dose-dependent manner. Reduction of trigramin resulted in a marked decrease in its ability to block vWF binding to human platelets.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chrysoptin is a potent glycoprotein IIb/IIIa fibrinogen receptor antagonist present in salivary gland extracts of the deerfly

Salivary gland lysates of the deerfly (genus Chrysops) contain chrysoptin, an inhibitor of ADP-induced platelet aggregation, which presumably assists the fly in obtaining a blood meal. Chrysoptin has now been isolated, and its cDNA has been cloned and expressed. Chrysoptin was purified to homogeneity using anion exchange and hydrophobic interaction chromatography and found to be a protein with a molecular mass of 65 kDa as determined by gel electrophoresis. N-terminal amino acid sequencing allowed for the synthesis of degenerate oligonucleotides that led to cloning, from salivary gland specific mRNA, of the cDNA encoding this platelet inhibitor. No RGD sites are present in the predicted sequence. A search of GenBank(TM) did not reveal significant sequence homology between chrysoptin and other proteins. The molecular mass predicted from the cDNA was 59 kDa. Predicted glycosylation and phosphorylation sites may account for this difference in molecular mass, as recombinant chrysoptin expressed in Sf21 cells had a molecular mass of 65 kDa, matching that of the natural protein. Chrysoptin functions by inhibiting the binding of fibrinogen to the fibrinogen/glycoprotein IIb/IIIa receptor on platelets with an IC(50) of 95 pmol. These results reveal that insect salivary glands are a source of fibrinogen receptor antagonists.     

Binding of glycoprotein IIIa-derived peptide 217-231 to fibrinogen and von Willebrand factors and its inhibition by platelet glycoprotein IIb/IIIa complex.

Based on previous reports in the literature and the high homology between platelet glycoprotein (GP) IIIa 217-231 and similar portions of other beta subunits of integrin receptors, we hypothesized that this region may participate in ligand binding. Using a polyclonal antibody against GPIIIa 217-231(YC), we tested the interaction of a synthetic peptide representing this region with fibrinogen (Fg), in the enzyme-linked immunosorbent assay (ELISA) system. Results show a calcium-independent, dose-related, direct interaction between GPIIIa 217-231(Y) and immobilized Fg. This peptide also bound to von Willebrand Factor (vWF) and fibronectin (Fn), but did not attach to a 50 kDa Fn fragment which is deficient in the cell attachment site. In addition, purified GPIIb/IIIa displaced GPIIIa 217-231(Y) from Fg and vWF. Binding of 125I-GPIIIa 217-231(Y) to Fg coated tubes was inhibited by soluble Fg and by the GPIIb/IIIa complex. We synthesized this peptide with several alterations; similar peptides with Pro-219 replaced with an Ala showed significantly reduced binding to Fg and vWF. The decreased binding of the peptides with Pro-219 substitutes suggests that the confirmation of GPIIIa 217-230 is important for its ability to bind to adhesive ligands. In conclusion, the amino acid residues between 217 and 231 of GPIIIa appear to be involved in ligand binding and Pro-219 probably plays a significant role in this interaction.     

The a1/a2 polymorphism of the glycoprotein IIIa gene and myocardial infarction in Caucasians with type 2 diabetes

A PlA1/A2 polymorphism of glycoprotein IIIa is known to be involved in the pathogenesis of coronary artery disease (CAD) and myocardial infarction (MI). The aim of this study was to investigate an association between the PlA1/A2 polymorphism of the glycoprotein IIIa gene and the risk of MI in Caucasians with type 2 diabetes. 549 Caucasians with type 2 diabetes were enrolled in the cross sectional retrospective case–control study: 224 patients with MI and 325 diabetic subjects without CAD. Blood biochemical analysis was performed. The polymerase chain reaction with restriction fragment length polymorphism was used for genetic analysis. Patients with MI were older (62 ± 11.8 vs. 58.5 ± 11.6 years; P < 0.001), and had a longer duration of type 2 diabetes (17.6 ± 8.9 vs. 15.1 ± 9.2; P = 0.01) compared to the diabetics without CAD. A significant difference in distribution of the A2A2 genotype of glycoprotein IIIa was not found between 224 diabetic patients with MI in comparison to 325 diabetics without CAD (11.6 vs. 14,1 %; n.s.). The diabetes duration and male sex were independent factors for the development of MI, whereas the PlA1/A2 polymorphism of glycoprotein IIIa was not. To conclude, The A2A2 genotype of the glycoprotein IIIa polymorphism was not associated with MI risk in Caucasians with type 2 diabetes.     

Morphological evidence for the association of plasma membrane glycoprotein IIb/IIIa with the membrane skeleton in human platelets

We examined the association between glycoprotein (GP) IIb/IIIa, a receptor for fibrinogen, and membrane skeletons in both unstimulated and thrombin-activated human platelets. After a treatment with dithiobis succinimidyl propionate (DTSP), a cross-linker, unstimulated and activated platelets were simultaneously extracted and fixed with a fixing solution containing Triton X-100. Also, the localization of GPIIb/IIIa on the plasma membrane was observed by a preembedding staining method of unextracted platelets. In unstimulated platelets, 20-40% of the whole plasma membrane remained in the detergent-extracted samples. Amorphous structures with 10-70 nm in diameters are distributed at 20 to 100-nm intervals on the surface of plasma membrane. Similar structures also were identified in the intact platelets by the immunocytochemical method. By careful inspection, we found that most of the amorphous structures that contained gold particles were connected to the submembrane zone just beneath the plasma membrane. The submembrane zone was identified as the membrane skeleton because actin was detected in the zone. After activation, detergent-insoluble granules were surrounded by dense networks of microfilaments in the central part of platelets. The filaments were identified as actin and became associated with myosin. These results demonstrate that GPIIb/IIIa on the plasma membrane is connected to the membrane skeleton and suggest that, during activation, actin filaments which extend into the cytoplasm from the membrane skeleton increase and form dense networks around Triton-insoluble granules.     

Morphological evidence for the association of plasma membrane glycoprotein IIb/IIIa with the membrane skeleton in human platelets

Summary We examined the association between glycoprotein (GP) IIb/IIIa, a receptor for fibrinogen, and membrane skeletons in both unstimulated and thrombin-activated human platelets. After a treatment with dithiobis succinimidyl propionate (DTSP), a cross-linker, unstimulated and activated platelets were simultaneously extracted and fixed with a fixing solution containing Triton X-100. Also, the localization of GPIIb/IIIa on the plasma membrane was observed by a preembedding staining method of unextracted platelets. In unstimulated platelets, 20–40% of the whole plasma membrane remained in the detergent-extracted samples. Amorphous structures with 10–70 nm in diameters are distributed at 20 to 100-nm intervals on the surface of plasma membrane. Similar structures also were identified in the intact platelets by the immunocytochemical method. By careful inspection, we found that most of the amorphous structures that contained gold particles were connected to the submembrane zone just beneath the plasma membrane. The submembrane zone was identified as the membrane skeleton because actin was detected in the zone. After activation, detergent-insoluble granules were surrounded by dense networks of microfilaments in the central part of platelets. The filaments were identified as actin and became associated with myosin. These results demonstrate that GPIIb/IIIa on the plasma membrane is connected to the membrane skeleton and suggest that, during activation, actin filaments which extend into the cytoplasm from the membrane skeleton increase and form dense networks around Triton-insoluble granules.     

N-terminal sequence of human leukocyte glycoprotein Mo1: conservation across species and homology to platelet IIb/IIIa

Mo1 and gp160-gp93 are two surface membrane glycoprotein heterodimers present on granulocytes and monocytes derived from humans and guinea pigs, respectively. We purified both antigens and found that their alpha subunits had identical N-termini which were significantly homologous to the alpha subunit of the human adhesion platelet glycoprotein IIb/IIIa.     

Case-Control Study of Platelet Glycoprotein Receptor Ib and IIb/IIIa Expression in Patients with Acute and Chronic Cerebrovascular Disease

Background

Animal models have been instrumental in defining thrombus formation, including the role of platelet surface glycoprotein (GP) receptors, in acute ischemic stroke (AIS). However, the involvement of GP receptors in human ischemic stroke pathophysiology and their utility as biomarkers for ischemic stroke risk and severity requires elucidation.

Aims

To determine whether platelet GPIb and GPIIb/IIIa receptors are differentially expressed in patients with AIS and chronic cerebrovascular disease (CCD) compared with healthy volunteers (HV) and to identify predictors of GPIb and GPIIb/IIIa expression.

Methods

This was a case—control study of 116 patients with AIS or transient ischemic attack (TIA), 117 patients with CCD, and 104 HV who were enrolled at our University hospital from 2010 to 2013. Blood sampling was performed once in the CCD and HV groups, and at several time points in patients with AIS or TIA. Linear regression and analysis of variance were used to analyze correlations between platelet GPIb and GPIIb/IIIa receptor numbers and demographic and clinical parameters.

Results

GPIb and GPIIb/IIIa receptor numbers did not significantly differ between the AIS, CCD, and HV groups. GPIb receptor expression level correlated significantly with the magnitude of GPIIb/IIIa receptor expression and the neutrophil count. In contrast, GPIIb/IIIa receptor numbers were not associated with peripheral immune-cell sub-population counts. C-reactive protein was an independent predictor of GPIIb/IIIa (not GPIb) receptor numbers.

Conclusions

Platelet GPIb and GPIIb/IIIa receptor numbers did not distinguish between patient or control groups in this study, negating their potential use as a biomarker for predicting stroke risk.     

The peptides APLHK, EHIPA and GAPL are hydropathically equivalent peptide mimics of a fibrinogen binding domain of glycoprotein IIb/IIIa

The anticomplementarity hypothesis predicted that the peptides APLHK, EHIPA, GAPL and LGVPPR would be functional mimics of a fibrinogen binding domain(s) in the fibrinogen receptor. The peptides APLHK and EHIPA were derived by translation of the cRNA of vitronectin m-RNA. The peptides GAPL and LGVPPR result from translations of the cRNA of von Willebrand factor m-RNA. The peptides APLHK, EHIPA, and GAPL, but not LGVPPRT, are hydropathically equivalent and inhibit fibrinogen binding to platelets. APLHK and EHIPA are hydropathic retromers. Thus for one pair of these peptides, the direction of their backbones did not affect function.     

Manipulation of chiral resolution for isoxazoline-based IIb/IIIa receptor antagonists using various mobile phase additives in subcritical fluid chromatography

Subcritical fluid chromatography (SubFC) using a carbon dioxide-methanol mobile phase is used for the chiral resolution of IIb/IIIa receptor antagonist enantiomers. The chiral resolution of three analogs, each containing two chiral centers, is optimized using various mobile phase additives. The effects that acidic, basic, and neutral additives have on retention, efficiency, and resolution are examined. The additive that gives the best resolution was found to be dependent upon the functionality and charge of the chiral analyte. For charged analytes, additives that act as competing ions of the same charge as the chiral analyte dramatically improve efficiency and resolution. Resolution of neutral chiral analyte enantiomers is also greatly affected by the choice of mobile phase additive. Chirality 10:338–342, 1998. © 1998 Wiley-Liss, Inc.     

Platelet glycoprotein IIIA PIA1/A2 polymorphism in young patients with ST elevation myocardial infarction and idiopathic ischemic stroke

Vacuolar protein sorting 4B (VPS4B), a member of ATPase family proteins, plays a crucial role in lysosome-dependent degradation. Recently, it was found that VPS4B could negatively regulate breast cancer progression through promoting lysosomal-dependent degradation for EGFR. Nevertheless, other studies found that VPS4B was also essential for cell division and mitosis through insuring the maintenance of centrosome and spindle assembly. Thus, the role of VPS4B in cancer biology remains under debate. In this study, we analyzed the clinical significance of VPS4B in NSCLC. The expression of VPS4B was evaluated by Western blot in 8 paired fresh NSCLC tissues and immunohistochemistry on 105 paraffin-embedded slices. VPS4B was highly expressed in NSCLC and significantly associated with NSCLCs tumor size, histological differentiation, clinical stage and Ki-67. Besides, high VPS4B expression was an independent prognostic factor for NSCLC patients’ poor survival. To determine whether VPS4B could regulate the proliferation of NSCLC cells, we knocked down the expression of VPS4B and analyzed the proliferation of A549 NSCLC cells using Western blot, CCK8 and flow cytometry assays, which together indicated that loss of VPS4B could inhibit cell cycle progress. These data suggest that VPS4B may promote the progression of NSCLC and be a biotarget for NSCLCs therapy.     

Atrial fibrillation after but not before primary angioplasty for ST-segment elevation myocardial infarction of prognostic importance

Aim

In patients with ST-segment elevation myocardial infarction (STEMI), it is uncertain whether atrial fibrillation has prognostic implications. There may be a difference between atrial fibrillation before and after reperfusion therapy.

Methods and results

In patients with STEMI treated with primary percutaneous coronary intervention (PCI), ECGs were analysed before and after primary PCI. Of the 1623 patients with electrocardiographic data before primary PCI, 53 patients (3.3%) had atrial fibrillation. Patients with atrial fibrillation were older, were more often female, and less often had anterior MI location. Of the 1728 patients with electrocardiographic data after primary PCI, 52 patients (3.0%) had atrial fibrillation. Atrial fibrillation was more common in older patients and in those with Killip class >1. Also patients with occlusion of the right coronary artery or TIMI flow 0 before primary PCI more commonly had AF after the procedure. Not successful reperfusion was also associated with a higher incidence of AF after primary PCI. Although both atrial fibrillation before and after primary PCI were associated with increased mortality, multivariable analyses, adjusting for differences in age, gender and Killip class on admission, revealed that atrial fibrillation after PCI (OR 3.69, 95% CI 1.87–7.29) but not before PCI (OR 1.86, 95% CI 0.89–3.90) was independent and statistically significantly associated with long-term mortality.

Conclusion

In patients with STEMI, atrial fibrillation after but not before primary PCI has independent prognostic implications. Possibly, atrial fibrillation after the PCI is a symptom of failed reperfusion and a sign of heart failure.