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PubMed2002

Expression and characterization of the ScFv fragment of antiplatelet GPIIIa monoclonal antibody SZ-21.

Platelet thrombus formation is a major contributor to various cardiovascular diseases caused by vascular occlusion. Glycoprotein IIb/IIIa (GPIIb/IIIa) plays a key role in platelet aggregation and hence the formation of thrombi. In the present study, the genes encoding the light- and heavy-chain variable regions (V(H) and V(L)) of SZ-21, which is a monoclonal antibody (MoAb) against GPIIIa integrin have been cloned by RT-PCR from the SZ-21 hybridoma. The genes of V(H) and V(L) were attached to the oligonucleotide of the linker peptide and single-chain antibody fragment (ScFv) was constructed. The ScFv was ligated into phage display vector pHEN1, and the phagemid pHEN1-21ScFv was constructed. The high-affinity phage display technology was used to retain the SZ-21ScFv binding activity to platelets in great effort. After four rounds of enrichment, the screening clone of SZ-21ScFv gene with good reactivity to platelets was ligated into highly expressed vector pET20b and expressed in Escherichia coli BL21(DE3)PlysS for the fusion protein. Recombinant ScFv fragment was produced mostly in the form of inclusion bodies, with its yield up to 21% of the total amount of bacteria protein. The ScFv fragment with the similar binding activity to platelets as MoAb SZ-21 was confirmed by enzyme-linked immunosorbent assay (ELISA) and Western blot. ADP-induced platelet aggregation can be inhibited by ScFv fragment in a dose-dependent manner and the maximal inhibition rate was obtained at a concentration of 750 nM. In addition, the ScFv fragment has ability to inhibit the binding of fibrinogen to platelets and react with endothelial cells. In this study, we have successfully produced the SZ-21ScFv, which retained the binding affinity to platelets and antithrombotic ability of their murine counterparts.

Animals↗
PubMed2002

Reductions in platelet contractile force correlate with duration of cardiopulmonary bypass and blood loss in patients undergoing cardiac surgery.

Blood loss secondary to platelet dysfunction is known to be increased when the duration of cardiopulmonary bypass (CPB) is prolonged. The ability to correlate alterations in platelet function with the duration of bypass and early postoperative blood loss, however, has remained elusive. Platelet contractile force, a novel measure of platelet-mediated clot retraction, is known to be reduced following cardiac surgery and blockade of platelet adhesion receptors. The aim of this study was to determine if alterations in platelet contractile force (measured using whole blood) correlated with the duration of CPB and early postoperative blood loss. Thirty patients were entered into a study designed to measure platelet function before, during, and after CPB. Platelet aggregometry and surface expression of CD42b and CD61 were also measured (using whole blood) in a subset of subjects (n=10) to further characterize the intrinsic structural and functional defects induced by CPB. Reductions in platelet contractile force had a significant correlation with duration of CPB (r=0.564; P=0.002) and early blood loss (r=0.545; P=0.003). Although decreases in platelet contractile force and aggregation both correlated with CPB time in the smaller subset of patients tested, only platelet contractile force correlated with decreases in CD42b, CD61 and blood loss. The results of this study suggest that prolongation of CPB is related to increasing degrees of platelet dysfunction and that reductions in platelet contractile force are related to decreases in platelet adhesion receptors and early postoperative blood loss.

Aged↗
PubMed2002

Low incidence of paradoxical platelet activation by glycoprotein IIb/IIIa inhibitors.

The human platelet antigen-1 (HPA-1, Pl(A)) polymorphism has been proposed to influence the inhibitory actions of abciximab. Thus, we hypothesized that this polymorphism might also be the cause for paradoxical activation of platelets by GPIIb/IIIa inhibitors. The effects of abciximab (1-10 microg/ml), tirofiban (3-30 nM), or eptifibatide (0.3-3 microg/ml) on basal and ADP (3 microM)-induced CD62P externalization were measured in n=62 healthy blood donors and n=177 patients with stable coronary artery disease. All subjects were genotyped for the human platelet antigen-1 (HPA-1, Pl(A)) polymorphism by GALIOS(R) and fluorescence correlation spectroscopy. Although a significant platelet hyperreactivity was observed in the patients, the HPA-1 genotype did not influence basal or ADP-induced CD62P expression. A moderate (twofold) stimulation of CD62P expression by abciximab but not by tirofiban or eptifibatide was observed in one patient. Interestingly, this patient carried the HPA-1 b/b genotype. In no other subject any activation of platelets by GP IIb/IIIa inhibitors was observed and there were no statistically significant differences between HPA-1 genotypes with respect to the effects of GP IIb/IIIa inhibitors on basal or ADP-stimulated CD62P expression. It is concluded that paradoxical platelet activation by abciximab is a rare (<2%) phenomenon. HPA-1 b/b genotype might be a contributing factor but clearly does not predict platelet activation by GP IIb/IIIa inhibitors.

Abciximab↗
PubMed2002

Thrombin activity and platelet microparticle formation are increased in type 2 diabetic platelets: a potential correlation with caspase activation.

Diabetics suffer from many complications including a prothrombotic condition. Activated platelet membrane provides an anchor, phosphatidylserine, for the attachment of the prothrombinase complex, which allows increased thrombin formation. This study aimed to further elucidate the interrelationship between coagulation proteins and activated platelets in type 2 diabetic blood. We found that there was a significant increase (30 x) in thrombin activity in the type 2 diabetic (ZDF) blood as compared to age-matched (ZL) controls (p<0.001). There was also a significant increase in the number of platelet microparticles in the type 2 diabetic rat compared to the lean control (p<0.001). Further, there were significant increases in caspase-3, -6, and -8 activities in the type 2 diabetic rats as compared to the lean controls (p<0.05). The combination of increased thrombin activity, increased PMP formation and increased caspase activity may contribute to the hypercoagulability of the diabetic blood. These results give more insight into the mechanisms underlying the interrelationship between diabetic platelets and coagulation proteins causing a prothrombotic condition in this patient population at increased risk from thromboembolic events.

Animals↗
PubMed2003

Increased prevalence of platelet glycoprotein IIb/IIIa PLA2 allele in ischaemic stroke associated with large vessel pathology.

INTRODUCTION: Platelet glycoprotein IIb/IIIa is a membrane receptor with a central function in the platelet adhesion and ultimately in the thrombus formation. Two major variants of the gene encoding the IIIa subunit, called PLA1 (A1) and PLA2 (A2), have been identified in the general population. There are indications that the A2 allele can also be associated with acute thrombosis or stroke. The purpose of this study was to study the distribution of the A2 allele in different vascular subtypes of stroke disease. MATERIALS AND METHODS: A total of 638 consecutive patients were analyzed and classified as having large vessel pathology (n=168) or a small vessel infarct (n=210). Localization of the vascular occlusions was deducted from analysis of the magnetic resonance imaging (MRI) scan results in stroke patients. The remainder patients were listed into a mixed vascular pathology group (n=167). Patients with other or poorly characterized stroke etiology were excluded from the study (n=93). RESULTS: In the small vessel and mixed vascular pathology groups, the PLA2 allele frequency was similar to that in the controls. By contrast, PLA2 allele frequency was approximately two-fold higher in patients with large vessel pathology (23.3%) than in the stroke-free control subjects (11.7%, p<0.0005). Multivariate logistic regression analysis of data confirmed this association with an odds ratio (OR) of 2.9 (95% confidence interval [CI]: 1.6-4.9, p<0.0005). CONCLUSIONS: These data suggest that the PLA2 allele is more frequent in brain infarcts associated with large-vessel occlusion.

Adult↗
PubMed2001

Loss of the ClC-7 chloride channel leads to osteopetrosis in mice and man.

Chloride channels play important roles in the plasma membrane and in intracellular organelles. Mice deficient for the ubiquitously expressed ClC-7 Cl(-) channel show severe osteopetrosis and retinal degeneration. Although osteoclasts are present in normal numbers, they fail to resorb bone because they cannot acidify the extracellular resorption lacuna. ClC-7 resides in late endosomal and lysosomal compartments. In osteoclasts, it is highly expressed in the ruffled membrane, formed by the fusion of H(+)-ATPase-containing vesicles, that secretes protons into the lacuna. We also identified CLCN7 mutations in a patient with human infantile malignant osteopetrosis. We conclude that ClC-7 provides the chloride conductance required for an efficient proton pumping by the H(+)-ATPase of the osteoclast ruffled membrane.

Adenosine Triphosphatases↗
PubMed1989

348 cases of suspected neonatal alloimmune thrombocytopenia.

Serological and clinical data were collected in 348 cases of suspected neonatal alloimmune thrombocytopenia (NAT). Of the 144 mothers who were Zwa-negative, 107 had Zwa antibodies--alone (94); with HLA antibodies (12); or with Bra antibodies (1). Antibodies were detected in 12 of the 204 Zwa-positive mothers as follows: anti-Bra (9), anti-Zwb (1), anti-Baka with HLA antibody (1), and blood group B isoagglutinins (1). The frequency of NAT due to Bra incompatibility (19%) was second to Zwa (78%). Zwa-NAT was clinically the more severe (14% had intracranial haemorrhages) and responded well to either maternal platelet transfusions or intravenous IgG. In Bra-NAT intracranial haemorrhages were not observed and most children recovered without specific therapy.

Adolescent↗
PubMed2003

Variation of the platelet glycoprotein IIIa PI(A1/A2) allele frequencies in the three ethnic groups of Singapore.

BACKGROUND: Polymorphisms of the glycoprotein IIIa receptor have been shown to be associated with differences in platelet aggregability. The PI(A2) variant of the polymorphism has been reported to be an inherited risk factor for acute coronary events. Although the allele frequency of this polymorphism is well documented in Caucasian populations, studies involving Asian Indians, Malays and Chinese are lacking. We studied 706 random male individuals to determine the genotypic distribution of this polymorphism in Singapore. METHODS: Male subjects included in this study were drawn from those undergoing routine annual medical examinations offered by their employers. Venous blood was obtained from these patients after an overnight fast and from which genomic DNA was extracted. Genotyping was carried out by polymerase chain reaction (PCR) followed by digestion with restriction enzyme NciI. Personal and family medical history of the subjects were also taken. RESULTS: The genotype distribution of the individuals studied was in accordance to a population at Hardy Weinberg equilibrium. The frequency of the PI(A2) allele was 0.1, 0.01 and 0.01 in the Indians, Malays and Chinese, respectively. The differences in frequencies of the PI(A2) variant are significant among different ethnic groups (P<0.001 for Indians vs. Chinese and Indians vs. Malays). CONCLUSIONS: We observed a significantly higher frequency of the PI(A2) allele among Indians relative to the Chinese and Malays in Singapore. The effect of this genotype may partially explain the higher rate of ischaemic heart disease seen among Indians compared to the Chinese and Malay ethnic groups.

Adult↗
PubMed2003

The disulfide-rich region of platelet glycoprotein (GP) IIIa contains hydrophilic peptide sequences that bind anti-GPIIIa autoantibodies from patients with immune thrombocytopenic purpura (ITP).

Immune thrombocytopenic purpura (ITP) is an autoimmune blood disease caused by autoantibody-mediated destruction of blood platelets. Platelet glycoprotein (GP) IIb/IIIa is a common target for antiplatelet autoantibodies. The present studies were undertaken (1). to confirm whether the disulfide rich repeat region of GPIIIa contains target epitopes for antiplatelet antibodies in patients with ITP; (2). to determine whether these antigens were defined by peptide sequences in the absence of post-translational modification; and (3). to correlate observed immunologic reactivity with the recently solved X-ray crystallographic structure of an analogous integrin complex, the vitronectin receptor, alpha(V)beta(3). Recombinant fusion proteins of four GPIIIa extracellular sequences were prepared and purified. Immunoblotting results with purified recombinant peptides showed potent reactivity of 16 of 24 ITP patient serum anti-GPIIb/IIIa antibodies with the fusion protein containing the GPIIIa sequence of residues from 468 to 691. These results are consistent with a report by Kekomaki et al. that a 50 kDa chymotryptic digestion product of GPIIIa isolated from blood platelets contains target epitopes for serum antiplatelet antibodies in 16 of 33 ITP patients. Smaller peptides including residues 446-501 and residues 593-691 each reacted with only 5 of the 24 patient sera; furthermore all but 3 of these interactions were very weak. Visualization of the conformation of the extracellular portion of alpha(V)beta(3) reveals the location of the 222-residue antigenic GPIIIa (beta(3)) peptide 'B' at the immediately extracellular region of the protein that includes a beta-tail domain and several integrin-EGF domains. In summary, predictions of hydrophilicity, surface accessibility and antigenicity and the three dimensional structure of the beta(3) integrin correlate with autoantibody binding to a recombinant GPIIIa peptide 'B' containing residues 468-691.

Amino Acid Sequence↗
PubMed1988

[Platelet phenotyping and study of alloantibodies using flow cytometry].

In this study, we describe a flow cytometric technic for the detection and characterization of platelet allo antibodies and for platelets grouping in the platelet group PLA. This new technique is a variant of the platelet suspension immuno fluorescence test. It is rapid, simple, sensitive and specific. So it is very useful in the cases of neonatal thrombocytopenia and posttransfusion purpura. Moreover, anti-HLA antibodies don't obstruct the detection of anti-PLA1 antibodies.

Antigens, Human Platelet↗
PubMed2002

Identification and characterization of a novel member of olfactomedin-related protein family, hGC-1, expressed during myeloid lineage development.

We have cloned a novel hematopoietic granulocyte colony-stimulating factor (G-CSF)-induced olfactomedin-related glycoprotein, termed hGC-1 (human G-CSF-stimulated clone-1). mRNA differential display was used in conjunction with a modified two-phase liquid culture system. Cultures were enriched for early precursors of erythroid, myeloid, and megakaryocytic lineages, which were isolated after induction with erythropoietin, G-CSF, and thrombopoietin, respectively. RNA from the enriched cells was subjected to differential display analysis to identify lineage-specific expressed genes. One clone specifically induced by G-CSF, hGC-1, was characterized. The 2861 bp cDNA clone of hGC-1 contained an open reading frame of 1530 nucleotides, translating into a protein of 510 amino acids with a signal peptide and six N-linked glycosylation motifs. The protein sequence of hGC-1 showed it to be a glycoprotein of the olfactomedin family, which includes olfactomedin, TIGR, Noelin-2 and latrophilin-1. Olfactomedin-like genes show characteristic tissue-restricted patterns of expression; the specific tissues expressing these genes differ among the family members. hGC-1 was strongly expressed in the prostate, small intestine, and colon, moderately expressed in the bone marrow and stomach, and not detectable in other tissues. In vitro translation and ex vivo expression showed hGC-1 to be an N-linked glycoprotein. The hGC-1 gene locus mapped to chromosome 13q14.3. Together, our findings indicate that hGC-1 is primarily expressed as an extracellular olfactomedin-related glycoprotein during normal myeloid-specific lineage differentiation, suggesting the possibility of a matrix-related function for hGC-1 in differentiation.

Amidohydrolases↗
PubMed1999

An ELISA for quantification of murine IgG in rat plasma: application to the pharmacokinetic characterization of AP-3, a murine anti-glycoprotein IIIa monoclonal antibody, in the rat.

An enzyme-linked immunosorbent assay (ELISA) has been developed to determine concentrations of murine IgG in rat plasma. Specifically, the assay was developed to measure a murine anti-glycoprotein IIIa antibody (AP-3) in rat plasma to facilitate future investigations of AP-3 pharmacokinetics and pharmacodynamics in the rat. The working range of the assay is 15-100 ng ml(-1), corresponding to a limit of quantification of 1.5 microg ml(-1) in rat plasma. The assay was validated with respect to accuracy, precision, and cross-reactivity with both pooled rat and mouse IgG. Intra-assay recoveries of AP-3 in rat plasma ranged from 93 to 103%, with CV% values ranging from 5.2 to 8.5%. Inter-assay recoveries of the plasma AP-3 samples ranged from 107 to 119% with CV% values ranging from 17.7 to 25.1%. The assay has no appreciable cross reactivity with pooled rat IgG and full cross reactivity with pooled mouse IgG, making this an ideal assay to determine plasma pharmacokinetics of mouse antibodies in the rat. The assay was used to determine the pharmacokinetics of AP-3 in a Sprague-Dawley rat.

Animals↗
PubMed2000

Glycoprotein IIIa Pl(A1/A2) polymorphism and sudden cardiac death.

OBJECTIVES: We studied the association of the Pl(A1/A2) polymorphism with coronary thrombosis, myocardial infarction (MI) and sudden cardiac death (SCD) in autopsied victims of sudden death. BACKGROUND: Sudden cardiac death is one of the leading symptoms of coronary heart disease in early middle age. Platelet glycoprotein (GP)IIb/IIIa fibrinogen receptors play a key role in coronary thrombosis and MI. Pl(A1/A2) polymorphism of the gene for GPIIIa has been previously studied in hospital MI patients. Significance of the Pl(A1/A2) polymorphism in victims of SCD is not known. METHODS: The Pl(A1/A2) polymorphism was studied in the Helsinki Sudden Death Study comprising 700 autopsied middle-aged white Finnish men (33 to 70 years, mean 53 years) who suffered sudden or violent out-of-hospital death. RESULTS: Prevalence of the A2 allele decreased with age in the series. This decrease was observed among victims of SCD (n = 281) but not in men who died violently (n = 258) or of other diseases (n = 127). Of SCD victims below 50 years, 39.7% were carriers of the A2 allele compared with 28.3% among men under 50 who died of other causes (odds ratio [OR] 2.5, p = 0.01). Men with acute fatal coronary thrombosis (n = 39) were more often (OR 3.4, p < 0.01) carriers of the A2 allele than were men (n = 242) with SCD in the absence of acute coronary thrombosis (48.7% vs. 24.4%, respectively). In addition, men with MI and recent or old thrombosis (n = 67) were more often (OR 3.6, p = 0.005) carriers of the A2 allele than were men (n = 123) with MI in the absence of thrombosis (44.8% vs. 20.3%, respectively). These associations were especially strong in men under 60. CONCLUSIONS: Our results suggest that the A2 allele of the Pl(A1/A2) polymorphism of GPIIIa is a major risk factor of coronary thrombosis and may be one important predictor of SCD in early middle age.

Adult↗
PubMed2003

Resistance in vitro to low-dose aspirin is associated with platelet PlA1 (GP IIIa) polymorphism but not with C807T(GP Ia/IIa) and C-5T Kozak (GP Ibalpha) polymorphisms.

OBJECTIVES: We investigated whether three platelet gene polymorphisms, Pl(A1/A2), C807T, and C-5T Kozak (encoding, respectively, for platelet membrane glycoproteins (GP) IIIa, GP Ia/IIa, GP Ibalpha), could contribute to the resistance to a low dose of aspirin (160 mg/day). BACKGROUND: Aspirin antiplatelet effect is not uniform in all patients, and the mechanism by which some patients are in vitro resistant to aspirin remains to be determined. However, it has been suggested that polymorphisms of platelet membrane glycoproteins might contribute to aspirin resistance. METHODS: Ninety-eight patients on aspirin (160 mg/day) for at least one month were enrolled. Aspirin resistance was measured by the platelet function analyzer (PFA)-100 analyzer; genotyping of the three polymorphisms was performed using a polymerase chain reaction-based restriction fragment-length polymorphism analysis. RESULTS: Using a collagen/epinephrine-coated cartridge on the PFA-100, the prevalence of aspirin resistance was 29.6% (n = 29). Aspirin-resistant patients were significantly more often Pl(A1/A1) (86.2%; n = 25) than sensitive patients (59.4%; n = 41; p = 0.01). Of the 29 patients, 25 were reevaluated after having taken 300 mg/day aspirin for at least one month. Only 11 patients still have nonprolonged collagen epinephrine closure time, and these were all Pl(A1/A1). No relation was found between resistance status and C-5T Kozak or C807T genotypes. CONCLUSIONS: Platelets homozygous for the Pl(A1) allele appear to be less sensitive to inhibitory action of low-dose aspirin. This differential sensitivity to aspirin may have potential clinical implications whereby specific antiplatelet therapy may be best tailored according to the patient's Pl(A) genotype.

Aged↗
PubMed2001

Integrin tyrosine phosphorylation in platelet signaling.

The beta 3 integrin cytoplasmic tyrosine (ICY) motif of alpha IIb beta 3 becomes tyrosine phosphorylated during platelet aggregation, causing Shc and myosin to interact with the beta-integrin cytoplasmic domain. Platelets from mice lacking beta 3 ICY motif tyrosines formed defective aggregates and poorly retracted clots, establishing integrin tyrosine phosphorylation as a key mediator of beta 3-integrin signals.

Adaptor Proteins, Signal Transducing↗