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PubMed2005

Gene frequencies of the HPA-1 to 6 and Gov human platelet antigens in Thai blood donors.

Human platelet alloantigens (HPA) are important in neonatal alloimmune thrombocytopenia (NAIT), posttransfusion purpura (PTP), platelet transfusion refractoriness, passive alloimmune thrombocytopenia, and transplantation-associated alloimmune thrombocytopenia. Thus, HPA genotyping is essential in diagnosis and treatment. We analyzed HPA-1 to 6 and Gov alleles, using PCR with sequence specific primers (PCR-SSP) in 500 Thai blood donors who had been HLA class I antigen typed. HPA-4a was present in all samples. HPA-1b, -2b, -5b, and -6b were rare, and HPA-4b was not found. HPA-3a and -3b showed frequencies of 56.0 percent and 44.0 percent, respectively. Gova and Govb showed frequencies of 49.1 percent and 50.9 percent, respectively. The prevalence rates of HPA-1 to 6 gene frequencies (GFs) were consistent with those of other Asian populations rather than those of Caucasians. We also report on the GFs of Gova and Govb, which also are comparable to those of Asian populations. Our results could establish a useful HPA- and HLA-matched plateletpheresis donor file and provide an improvement of platelet alloantibody detection in alloimmune thrombocytopenic patients, and, therefore, a more effective platelet transfusion program.

Adult↗
PubMed2005

Is platelet function as measured by Thrombelastograph monitoring in whole blood affected by platelet inhibitors?

Platelet inhibitors, especially the glycoprotein (GP) IIb/IIIa receptor antagonists, have demonstrated their effectiveness in reducing the acute ischemic complications of percutaneous coronary intervention (PCI) and in improving clinical outcomes in patients with acute coronary crisis. Three common platelet inhibitors observed in emergent cardiopulmonary bypass (CPB) for failed PCI are abciximab, eptifibatide, and tirofiban. An in vitro model was constructed in two parts to determine whether platelet aggregation inhibition induced by platelet inhibitors would be demonstrated by the Thrombelastograph (TEG) monitor when compared with baseline samples with no platelet inhibitor. In part A, 20 mL of fresh whole blood was divided into four groups: group I = baseline, group A = abcix-imab microg/mL, group E = eptifibatide ng/mL, and group T = tirofiban ng/mL. Platelet inhibitor concentrations in whole blood were derived starting with reported serum concentrations with escalation to achieve 80% platelet inhibition using the Medtronic hemoSTATUS and/or Lumi-aggregometer. A concentration range determined by our in vitro tests were chosen for each drug using concentrations achieving less than, equal to, or greater than 80% platelet inhibition. In part B, TEG analysis was then performed using baseline and concentrations for each drug derived in part A. Parameters measured were clot formation reaction time (R), coagulation time (K), maximum amplitude (MA) and alpha angle (A). Groups E1000 and E2000 extended R over control by 37% and 23%, respectively (p = 0.01 and 0.03). Groups E1000 and E2000 increased K times by 45% and 58% (p = .02 and .04). T160 samples prolonged K by 20% (p = 0.01). The angle or clot strength (A) was decreased in groups T160 and E1000 by 23% (+ 7.06 SD) and 18% (+ 11.23 SD), respectively (p = 0.001 and 0.01). The MA decrease was statistically significant in the T160, E1000 and E2000 by 9%, 6% and 13% respectively (p = 0.01). Samples treated with abciximab were comparable to control values for all parameters measured. Although statistical significance could be demonstrated with some parameters, sensitivity was only observed at increased doses and was not seen with all agents tested. In our in vitro model, the TEG monitor was unable to demonstrate clinically significant differences in platelet function and may not be reflective of platelet function in samples which have been treated with these GP IIb/IIIa inhibitors.

Abciximab↗
PubMed2005

Fetomaternal alloimmune thrombocytopenia presenting as intracerebral bleeding in utero.

Feto-maternal alloimmune thrombocytopenia is a serious fetal disorder resulting from platelet antigen incompatibility between the mother and the fetus. Intracranial bleeding is the most serious complication of alloimmune thrombocytopenia and can result in severe disability and death in utero. The authors report a case of intracerebral hemorrhage in utero resulting from alloimmune thrombocytopenia.

Adult↗
PubMed2005

[Results of clinical trials of a novel glycoprotein IIb-IIIa antagonist framon in high-risk coronary angioplasty].

New glycoprotein (GP) IIb-IIIa antagonist preparation framon (Monafram), is the F(ab')(2) fragment of a monoclonal antibody FRaMon directed against GP IIb-IIIa. This preparation blocks GP IIb-IIIa binding with fibrinogen and inhibits platelet aggregation both in vitro and upon intravenous administration. Safety and ability of framon to prevent thrombotic complications in high risk coronary angioplasty (CA) was evaluated in the present study. FRAMON was injected intravenously into 153 patients just before the start of procedure as a single bolus at the dose of 0.25 mg/kg. Control group was formed of 126 patients who underwent angioplasty without GP IIb-IIIa blockers. After framon administration there were no allergic reactions or major bleedings, deep thrombocytopenia (< 50000/microl) developed in 1 patient (< 1%), and antibodies against framon were detected in less than 5% of patients. Number of unfavorable outcomes (cardiovascular death, myocardial infarction, angina recurrence) within 1 month after CA was 3 times higher in control group than in the group of patients treated with framon (11.4% and 3.3%, respectively, p = 0.018). The effect of framon was most strongly pronounced within the first day after procedure -- administration of the drug reduced number of acute thromboses from 6.5% to 0.7% (p = 0.013). Significant differences between numbers of end points was still preserved at 6 months after procedure (25.7 and 14.2% in control and framon groups, respectively, p = 0.023). The data obtained proved safety and clinical efficacy of framon administration in coronary angioplasty with high risk of thrombotic complications.

Angioplasty, Balloon, Coronary↗
PubMed2005

[Novel mutation of the GPIIIa gene in the Russian population, Leu40Arg, linked to the Leu33Pro].

Glycoprotein IIb/IIIa complex, a platelet surface fibrinogen receptor, plays a key role in producing primary hemostasis. At present, only a single mutation in the GPIIla gene, Leu33Pro, and a single mutation in the GPIIb gene, lle843Ser, has been described. The mutations are known to enhance signaling functions of the receptor and are associated with the development of arterial thromboses. In the present study, we describe a novel GPIIIa mutation, which is T to G nucleotide substitution in position 1585, resulting in the replacement of Leu for Arg in position 40 of the amino acid sequence of the protein.

Adolescent↗
PubMed2005

Comparative study of myelodysplastic syndromes and normal bone marrow biopsies with conventional staining and immunocytochemistry.

OBJECTIVE: To study the histomorphometric features of megakaryocytic elements in bone marrow trephine biopsies of various subtypes of myelodysplastic syndrome (MDS). STUDY DESIGN: Comparative image morphometry using hematoxylin-eosin-stained and CD 61-stained trephine biopsies was carried out on 40 cases of MDS and 10 normal subjects to analyze the megakaryocytes objectively. The various variables analyzed were number of megakaryocytes and micromegakaryocytes, area, perimeter and diameter of the megakaryocytic elements. RESULTS: The mean number of megakaryocytes was lower in cases of MDS as compared to the normals in all except for a single case of hypoplastic MDS, in which the megakaryocytes were more abundant (3.6 per high-power field [hpf]). No micromegakaryocytes were observed in the 2 cases of chronic myelomonocytic leukemia. The mean area, perimeter and diameter of megakaryocytes decreased significantly on immunostaining with CD 61. CONCLUSION: The mean number of megakaryocytes per hpf was lower in the cases of MDS as compared to normal cases on hematoxylin-eosin. However, on CD 61 staining the number of megakaryocytes per hpf increased in cases of MDS. Micromegakaryocytes were seen in scanty numbers in the normals but increased in MDS cases and increased significantly on CD 61 immunostaining. The mean area, perimeter and diameter of megakaryocytes decreased significantly on immunostaining with CD 61, indicating the increased numbers of micromegakaryocytes in MDS. Hence, immunostaining is an efficient method of detecting increased numbers of megakaryocytes and micromegakaryocytes that would ordinarily be missed by using routine hematoxylin-eosin staining.

Anemia, Aplastic↗
PubMedDate not supplied

[Prognostic implications of GP3a glucoprotein gene PLA1/PLA2 allele in prostatic cancer: pilot results of the study].

We studied the role of integrins, primarily, the role of allele distribution of GP3a gene in development of prostatic cancer (PC) and assessment of its prognostic significance. From November 2003 to May 2004 we examined 32 patients with PC: 11 patients with local PC T1-2N0M0; 14 patients with locally advanced cancer T3N0M0 and 7 patients with invasive and/or metastatic cancer T3-4N10-1 or T3-4N0-1M1. The blood from all the patients we studied with PCR for alleles of GP3a gene, PSA. Seventeen patients were found to have alleles PLA1A1, 14(44%)--alleles PLA1A2, 1(3%)--alleles PLA2A2. Alleles PLA1A2 occurred significantly more often than in the population (p < 0.005). The group analysis has found that 8 patients with local PC had alleles PLA1A1, 3 patients--alleles PLA1A2 (27%). We discovered alleles PLA2A2, PLA1A1 and PLA1A2 in 1(7%), 5(36%) and 8(57%) patients with locally advanced PC, respectively. Among patients with metastatic and/or invasive prostatic cancer, there were 4 (57%) and 3 (43%) cases of alleles PLA1A1 and PLA1A2, respectively. Our study demonstrated influence of carriage of PLA2 allele on occurrence of PC risk (5-fold higher) and its invasive forms (10-fold higher and more). Probability to develop local invasion among patients with prostatic cancer--carriers allele PLA1A2 is 6 times higher than among carriers of alleles PLA1A1. A PC course in carriers of alleles PLA1A2 may be characterized by faster development of local invasion and metastasizing vs carriers of alleles PLA1A1. These findings can be used in design of nomograms for prognostication of invasion of clinically small tumors in verification of significance on greater number of the patients.

Aged↗
PubMed2005

Is there a relationship between anti-HPA-1a concentration and severity of neonatal alloimmune thrombocytopenia?

There is uncertainty about the relationship between anti-HPA-1a levels and severity of neonatal alloimmune thrombocytopenia (NAIT). To investigate this relationship further,the concentration of anti-HPA-1a in HPA-1b homozygous women was determined, using a newly developed quantitative ELISA that uses purified anti-HPA-1a to obtain a standard curve. Seventy-eight samples collected from 22 HPA-1b homozygous pregnant women at various stages of pregnancy were tested. These included five women who had delivered babies with severe NAIT. A national HPA-1a antibody standard (NIBSC 93/710), designated as 1 arbitrary unit/mL (AU/mL),was used in each ELISA to calibrate the purified anti-HPA- 1a, enabling the presentation of results as AU/mL. Moreover, selected samples were also assayed by PAK 12 and their reactivity compared with quantity of antibody. The use of the purified HPA- 1a antibody yielded consistent sigmoid curves, enabling the measurement of HPA-1a antibody concentration in the test samples. The antibody concentration was significantly correlated with the antibody titer in the 78 samples studied (R = 0.54, p < 0.001). Furthermore, there was a significant correlation between PAK 12 and the quantitative ELISA in a selected number of cases, with or without NAIT (R = 0.71, n = 10; p < 0.02). On the other hand, there was no correlation of antibody concentration with NAIT incidence (R = -0.046). This study indicates that there is no relationship between anti-HPA-1a concentration and severity of NAIT when ELISA is used, although the correlation between ELISA and other methods, such as monoclonal antibody immobilization of platelet antigens (MAIPA) assay, remains to be determined.

Antigens, Human Platelet↗
PubMed2005

[A new method for the detection of platelet derived microparticles by cytometric bead array and its evaluation].

OBJECTIVE: To establish a new method for the detection of platelet derived microparticles (PMPs) by cytometric bead array and to evaluate the characteristic of this method. METHODS: Using detection beads coated with monoclonal antibody against platelet glycoprotein IIIa to capture PMPs, then using CD41 PE to react with PMPs captured by detection beads, finally using flow cytometer detect beads-PMPs-CD41 PE complex. Anticoagulant, reaction time and other condition were optimized. Then reproducibility and linear range of this method were evaluated. PMPs of 45 health adults were determined by this method. RESULTS: Detection beads were stable in 4 degrees C for over 90 days (coefficient of variance was 0.65%) which matched the basic request of clinical detection. CTAD could restrain the in vitro release of PMPs efficiently; The reaction could reach maximum combination at room temperature after 4 hour; Non-specific absorbance of detection beads could be eliminated by washing of PBS. This method had preferable reproducibility (CV in batch was 6.8%; CV between batch was 10.4%) and a wide linear range from 0 to 200 microl of platelet activation suspension (r = 0.9962). PMP level of 45 health adults was 1.031-1.766. CONCLUSION: PMPs can be detected exactly by cytometric bead array, and it's important for PMPs detection to be an assistant for diagnosis of haemorrhagic and thrombotic diseases.

Adult↗
PubMedDate not supplied

Plasminogen activator inhibitor type 1 (PAI-1) and platelet glycoprotein IIIa (PGIIIa) polymorphisms in young Asian Indians with acute myocardial infarction.

BACKGROUND: The relationship between polymorphisms in the genes for plasminogen activator inhibitor type 1(PAI-1) and platelet glycoprotein IIIa (PGIIIa), clinical and environmental features, and the risk of premature coronary heart disease (CHD) in Asian Indian subjects living in South Africa, has been investigated. METHODS: The prevalence of the PAI-1 promoter 4G/5G and the PGIIIa PI A1A2 polymorphisms was examined in 195 unrelated Asian Indian patients ( <or= 45 years) who presented with myocardial infarction (MI). Results were compared with those from 107 unaffected siblings (18-45 years) and 300 healthy age- and race-matched control subjects. RESULTS: Overall, neither the PAI-1 4G/5G nor the PGIIIa PI A1A2 polymorphism demonstrated an independent risk for MI. No synergistic effect was observed between these two polymorphisms when analysed together. There was a marginal association between the 4G allele of the PAI-1 gene and the risk of MI in individuals who smoked compared with non-smokers (26 vs 11%; p = 0.028; OR 2.74; 95% CI 1.04-8.47). The PGIIIa PI A2 allele was, however, strongly associated with a previous history of MI (17 vs 6%; p = 0.004; OR 3.00; 95% CI 1.38-6.46), as well as the severity of disease as determined by angiography (single/double- vs triple-vessel disease: 3% vs 15%; p = 0.020; OR 0.19; 95% CI 0.02-0.92). CONCLUSION: In young Asian Indians who smoke, the PAI-1 4G allele is a mild risk factor for the development of MI. The PGIIIa PI A2 allele constitutes a significant risk for individuals who have a previous history of MI, as well as serving as an indicator for the severity of CHD.

Adolescent↗
PubMed2005

Polymorphism of the glycoprotein Ia and IIIa in the group of women in childbirth does not correlate with an increased risk of developing thrombosis.

OBJECTIVE: The aim of the study was to evaluate the prevalence of the GP Ia and GP IIIa polymorphisms in the group of women in labor, and to assess the risk of thrombosis associated with their occurrence. DESIGN AND SETTINGS: 245 women in labor hospitalized between 1.01.2001 and 31.12.2003 r. were enrolled in the study. Patients were qualified for the study if detailed physical exam and past medical history excluded existence of known risk factors predisposing to thrombosis. Study group was composed of 72 women in childbirth, which at some point during current pregnancy or in early labor were diagnosed with thrombosis, and control group included 173 women in labor randomly picked from the group of patients with uncomplicated pregnancies. Polymorphic regions of platelets glycoprotein were detected using genotyping methods based on polymerase chain reactions (PCR). RESULTS: 1.72% of patients were found to have thrombosis. The thrombosis was located in the venous system in 97.2% of cases. Arteries were affected in two patients (2.7%). Prevalence of individual platelets glycoprotein mutations did not differ between controls and study group. In both groups platelets glycoprotein polymorphisms moderately pro-thrombotic A1/A2 and C/T dominated, and the least numerous were strongly pro-thrombotic A2/A2 and T/T. CONCLUSIONS: Our results did not show the causative relationship between the existence of platelets GP IIIa/GP Ia mutations and venous system thrombosis in the women in labor. Probably presence of pro-thrombotic mutations of platelets glycoprotein in this group of patients does not represent the increased risk of thrombosis.

Adult↗
PubMed2006

[The immunoreactivity of IgG and its fragments from ITP patients and their effects on platelet aggregation function].

OBJECTIVE: To prepare ITP plasma IgG and its F(ab')2 fragments and investigate their immunoreactivity to platelet GPIIb/IIIa and/or GPIb/IX and their effects on platelet aggregation function. METHODS: The ITP patients having inhibitory autoantibody to the platelet aggregation were selected by modified MAIPA and platelet aggregation test with turbidimetry. Plasma IgG and its F(ab')2 fragments were prepared by streptococcal protein A affinity column and pepsin digestion. The immunoreactivity and the effects on platelet aggregation function of the whole antibody and its fragments were detected by modified MAIPA and platelet aggregation test, respectively. RESULTS: (1) Anti-platelet GPIIb/IIIa and/or GPIb/IX autoantibodies were detected in 34 of 68 (53.6%) ITP patients' plasmas and that from 5 patients significantly inhibited the platelet aggregation induced by ADP or ristocetin. (2) By using protein A column combined with protease digestion, pure IgG and its F(ab')2 fragments were successfully obtained. (3) The purified IgG and its F(ab')2 fragments retained the ability to bind to their respective glycoproteins and inhibited the platelet aggregation function, whereas the IgG depleted plasma lost the ability of binding to the platelet GPs. CONCLUSIONS: F(ab')2 fragment of the IgG antibody is a functional fragment, which not only has the binding ability to the platelet GPs but also inhibits the platelet aggregation function in a dose-dependent manner.

Adolescent↗
PubMed2006

[Effect of anti-Helicobacter pylori ureB monoclonal antibody on platelet aggregation and activation, and its mechanism study].

OBJECTIVES: To study the effect of monoclonal antibody (McAb) against helicobacter pylori (Hp) ureB, 1F11 on platelet aggregation and activation, and its mechanism. METHODS: The relativity between human platelet glycoproteins (GPs) and Hp ureB was identified by Western blot and FCM. Platelet aggregation was measured by turbidimetry, and P-selectin and TXB2 assay by ELISA. RESULTS: 1F11 could bind to platelet GPIIIa, and ADP-induced platelet aggregation was inhibited by 1F11 in a dose-dependent manner. However, 1F11 had no effect on plasma P-selectin and TXB2 induced by ADP. The FCM results show that the positive rates of platelet binding to FITC-SZ21 was decreased from 99.5% to 77.4% after addition of 1F11. CONCLUSION: McAb against Hp ureB 1F11 inhibits platelet aggregation through binding to platelet GPIIIa but does not block platelet activation. There might be crossed-epitopes on Hp ureB and platelet GPIIIa, and Hp infection might be involved in ITP immunopathology.

Antibodies, Bacterial↗
PubMed2006

[Occurrence of plasmic and platelet prothrombotic polymorphisms in women in childbirth].

DESIGN: Plasmic pro-thrombotic factors as well as pro-thrombotic platelet glycoprotein mutations have been shown to play an important role in the mechanism of the thrombo-embolic disease. However, there is no published study evaluating the role of above mentioned genetic factors in the thrombo-embolic episodes in women in childbirth. OBJECTIVE: The aim of the study was to evaluate the role of selected genetic factors in appearance of thrombo-embolic complications in the women in childbirth, and to determine if there is the coexistence of selected platelet glycoprotein polymorphisms and factor V Leiden mutations. MATERIAL AND METHODS: 71 cases of women in child birth with thrombo-embolic disease were analyzed. Selected demographic characteristics, and genetic pro-thrombotic factors like factor V Leiden mutation, and pro-thrombotic platelet glycoprotein GP Ia, and GP IIIa polymorphisms were examined. RESULTS: Amongst pro-thrombotic platelet glycoprotein polymorphisms moderately pro-thrombotic heterozygous A1/A2, and heterozygous C/T were most prevalent. The least common were strongly pro-thrombotic homozygous A2/A2, and T/T. Analysis of the factor V Leiden mutation revealed statistically significant difference in the presence of allele A, which determines the prothrombotic tendencies in its carrier. CONCLUSIONS: Our study shows that factor V Leiden mutation, and investigated platelet GP Ia, and GP IIIa polymorphisms frequently coexist. Moreover, presence of factor V Leiden mutation is a risk factor for thrombo-embolic disease in the women in childbirth.

Adult↗
PubMed2006

Type II Glanzmann thrombasthenia in a compound heterozygote for the alpha IIb gene. A novel missense mutation in exon 27.

BACKGROUND AND OBJECTIVES: Glanzmann thrombasthenia is an autosomal recessive bleeding disorder characterized by a life-long hemorrhagic tendency and absent or severely reduced platelet aggregation in response to agonists, caused by quantitative or qualitative abnormalities in the platelet fibrinogen receptor, integrin alphaIIb beta3. The aim of this study was to identify the molecular genetic defect and determine its functional consequences in a patient with type II Glanzmann thrombasthenia. DESIGN AND METHODS: The expression of platelet alphaIIb beta3 was determined by flow cytometry and western blotting. Mutations were identified by sequencing both cDNA and genomic DNA. Functional characterization was assessed by exontrap and transient transfection analysis. RESULTS: Flow cytometry and western blot analysis revealed markedly reduced levels of platelet alphaIIb beta3, which may account for the residual fibrinogen binding detected upon platelet activation. Sequencing of genomic DNA revealed the presence of two mutations in the alphaIIb gene: a C1750T transition in the last codon of exon 17 changing Arg553 to STOP, and a C2829T transition in exon 27 that changes Pro912 to Leu. Sequence analysis of reversely transcribed alphaIIb mRNA did not detect cDNA from the C1750T mutant allele, and revealed a significant increase of the physiological splicing out of exon 28 in the cDNA carrying the C2829T mutation. Transient expression of [912Leu]alphaIIb in CHO-b3 cells showed a marked reduction in the rate of surface expression of alphaIIb beta3. INTERPRETATION AND CONCLUSIONS: The results suggest that the thrombasthenic phenotype is the result of reduced availability of alphaIIb-mRNA, enhanced expression of exon 28-deleted transcripts, and defective processing of [912Leu]alphaIIb.

Animals↗
PubMed1990

[Use of a microtest plate method in pregnancy thrombocyte serology].

In search of a time saving concept for verifying a neonatal allo-immune thrombocytopenia (NATP) we tested the suitability of a solid phase platelet assay for antibodies (ab) which might induce NATP. Compared to PSIFT the test is equally appropriate for the detection of platelet specific and HLA ab. Using maternal, paternal, P1A1 neg. and random platelets the test gives quick answers to: a) is one dealing with a NATP? b) is anti-P1A1 involved?

Antigens, Human Platelet↗
PubMed1990

[219 Zw(a) positive mothers of children with clinically suspected neonatal alloimmune thrombocytopenia].

The sera of 219 Zwa-positive mothers who gave birth to children with clinically suspected neonatal alloimmune thrombocytopenia (NAIT) were tested for platelet-reactive antibodies using the platelet adhesion immunofluorescence test and a glycoprotein-specific immunoassay (MAIPA). In 102 families a serological crossmatch of maternal serum against paternal platelets was performed. 12 sera contained platelet specific antibodies: Zwb (1), Bra (4), Bra and HLA (5), Baka and HLA (1), Sra (1); three further sera contained antibodies against blood group A (1), blood group B (1), blood group B and HLA (1). Anti-Bra was the most important antibody in this cohort. There is evidence that in certain cases antibodies against blood group antigens A or B may cause NAIT. Clinical data of NAIT duo to anti-Bra suggest a less severe bleeding tendency than in NAIT due to anti-Zwa. The HLA-DRw6 antigen was identified as an immunogenetic marker for the immunization against the Bra-antigen.

Antibody Specificity↗
PubMed1991

A 13-mer peptide straddling the leucine33/proline33 polymorphism in glycoprotein IIIa does not define the PLA1 epitope.

We confirm the recent report (J Clin Invest 83:1778, 1989) of a polymorphism at amino acid 33 of platelet GPIIIa associated with the PLA1/PLA2 phenotype by using the polymerase chain reaction on cDNA derived from platelet RNA, using the base-pair primers 105-129 and 452-428. Platelet cDNA from three PLA2-homozygous individuals, when digested with Nci I, gave two bands of 256 bp and 91 bp, whereas eight PLA1 cDNAs gave a single band of 347 bp. Two 13-mer amino acid peptides straddling the amino acid polymorphism: SDEALP (L/P) GSPRCD were synthesized for epitope studies. Two mouse polyclonal antibodies were raised: one against the PLA1-associated peptide, the other against the PLA2 peptide. Both antibodies react with either peptide, as well as with both PLA1 and PLA2 platelets. The PLA1 peptide did not block the binding of two different human anti-PLA1 antibodies to the 100-Kd GPIIIa band on immunoblot of platelet extracts; neither did it block the binding of the same antibodies to PLA1-platelet extracts in an enzyme-linked immunosorbent assay. Further studies were performed on the PLA1 epitope following subtilisin digestion of purified GPIIIa. A 55-Kd fragment was obtained that retained the PLA1 epitope as well as the first 13 N-terminal amino acids of GPIIIa. Reduction of the 55-Kd fragment resulted in loss of the PLA1 epitope with production of a 67-Kd, 21-Kd, and 10-Kd band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The 55-Kd band does not react with LK-2, a monoclonal antibody versus GPIIIa that inhibits adenosine diphosphate, collagen, epinephrine, and thrombin-induced aggregation. Thus, the PLA1 epitope is conformation-induced, resides on an N-terminal 55-Kd fragment composed of two or more peptides held together by -SH bonds, and is not required for platelet aggregation.

Amino Acid Sequence↗