Platelet cDNA Research Articles

Rap2B GTPase: structure, functions, and regulation.

Rap2B GTPase, a member of Ras-related protein superfamily, was first discovered from a platelet cDNA library in the early 1990s. Since then, it has been reported to play an important role in regulating cellular processes including cytoskeletal organization, cell growth, and proliferation. It can be stimulated and suppressed by a wide range of external and internal inducers, circulating between GTP-bound active state and GDP-bound inactive state. Increasing focus on Ras signaling pathway reveals critical effects of Rap2B on tumorigenesis. In particular, Rap2B behaves in a p53-dependent manner in regulation of apoptosis and migration. Apart from being an oncogenic activator, Rap2B has been found to participate in many other physiological events via diverse downstream effectors. In this review, we present recent studies on the structure, regulation, and multiple biological functions of Rap2B, shedding light on its potential status in treatment of cancer as well as other diseases.

Fatal Bleeding Caused by Congenital Factor XIII Deficiency and Development of An Inhibitor in a Hispanic Male

We report a case of fatal bleeding in a patient with mild congenital Factor XIII deficiency who also developed an inhibitor that interfered with fibrin stabilization. Plasma Factor XIII circulates as a tetramer composed of two A-chain and two B-chains and is bound to fibrinogen. After thrombin cleaves the A-chain at Arg 37, the B-chain dissociate producing Factor XIIIa. Factor XIIIa catalyzes covalent linkages between the fibrin molecules aligned in the fibrin clot. Factor XIIIa makes the fibrin resistant to disruption by urea and plasmin. Factor XIIIa levels greater than 1 % are needed to covalently stabilize a clot. We studied a 39 year old Hispanic male that presented with altered mental status, headache and a large left frontal parietal hemorrhage. Ultimately, he became non-responsive and was intubated. He had no major bleeding history but did have frequent nosebleeds. Routine blood counts and coagulation laboratory tests were normal. However, a qualitative Factor XIII assay was abnormal and the clot dissolved in 5M Urea. Furthermore, a 1:1 mixing study did not correct the defect suggesting the presence of an inhibitor. A small dose of cryoprecipitate was administered to the patient and this corrected the clot stability defect. Four days later, the defect recurred and 150 ml of cryoprecipitate was administered and despite correction of the fibrin stabilizing abnormality the patient died. Two of his sisters had a history of repeated hemorrhagic miscarriages in Mexico a finding consistent with congenital Factor XIII deficiency. DNA sequencing of the factor XIII a-chain gene was performed from the propositus' mononuclear cells. A reverse transcription was done using an oligo(dT) 12–18 primer followed by nested PCR amplification and a heterozygous missense mutation was observed at codon 35 (G226T; Val35Leu). This mutation was than confirmed in propositus' platelet cDNA. The Val35Leu substitution is close to the thrombin cleavage site, the G226T substitution is in the catalytic core domain. This case demonstrates that in some cases of Factor XIII deficiency there is residual Factor XIIIa activity in the mutant molecule that can prevent serious bleeding. However, patients may develop autoantibodies that further interfere with Factor XIII function and may suffer serious bleeding complications. Additional cases of Factor XIII deficiency may exist in patients that have mild bleeding problems.

MPL mutations in myeloproliferative disorders: analysis of the PT-1 cohort

AbstractActivating mutations of MPL exon 10 have been described in a minority of patients with idiopathic myelofibrosis (IMF) or essential thrombocythemia (ET), but their prevalence and clinical significance are unclear. Here we demonstrate that MPL mutations outside exon 10 are uncommon in platelet cDNA and identify 4 different exon 10 mutations in granulocyte DNA from a retrospective cohort of 200 patients with ET or IMF. Allele-specific polymerase chain reaction was then used to genotype 776 samples from patients with ET entered into the PT-1 studies. MPL mutations were identified in 8.5% of JAK2 V617F− patients and a single V617F+ patient. Patients carrying the W515K allele had a significantly higher allele burden than did those with the W515L allele, suggesting a functional difference between the 2 variants. Compared with V617F+ ET patients, those with MPL mutations displayed lower hemoglobin and higher platelet levels at diagnosis, higher serum erythropoietin levels, endogenous megakaryocytic but not erythroid colony growth, and reduced bone marrow erythroid and overall cellularity. Compared with V617F− patients, those with MPL mutations were older with reduced bone marrow cellularity but could not be identified as a discrete clinicopathologic subgroup. MPL mutations lacked prognostic significance with respect to thrombosis, major hemorrhage, myelofibrotic transformation or survival.

Heterogeneous JAK2V617F Allele Burden in Essential Thrombocytosis: Gender and Clinical Correlates.

Essential thrombocytosis (ET) is the most heterogeneous of the chronic myeloproliferative disorders (MPD) when assessed by clonality assays, PRV-1, Mpl expression and JAK2V617F status. The purpose of this study was to prospectively analyze a cohort of 68 ET patients to determine the relationship of quantitative JAK2V617F allele percentage to disease features. The cohort consisted of 18 men and 50 women followed at our institution with a median disease duration of 7 years (range 1–24 years); all patients were clinically assessed between 2005 and 2006. Neutrophil genomic DNA and platelet cDNA were prepared from blood samples obtained at the time of clinical assessment and quantitative JAK2V617F measurements were made using a sensitive allele-specific, quantitative PCR assay. Median patient age at diagnosis was 47 years (range 2–84) without gender difference. JAK2V617F-positive patients were older than patients without the mutation (57 versus 41 years, p=<0.001). When assessed by neutrophil genomic DNA, more male than female ET patients were JAK2V617F-positive (56% versus 36%); however, when assessed by platelet cDNA, males and females had a similar JAK2V617F prevalence (60% versus 73%). JAK2V617F-positive male ET patients also had a higher median neutrophil allele percentage than JAK2V617F-positive female patients (47%, range 17–63, versus 31%, range 5–64). In 25 ET patients (15 female and 10 male), simultaneous JAK2V617F allele assessment in neutrophils and platelets identified 4 patients, all female, who were JAK2V617F-positive only in their platelet cDNA. JAK2V617F status did not correlate with the prevalence of erythromelalgia (4/11 JAK2V617F-positive), transient ischemic attack (3/7 JAK2V617F-positive), stroke (1/4 JAK2V617F-positive), migraine (7/18 JAK2V617F-positive), acute coronary syndromes (2/6 JAKV617F-positive), deep venous thrombosis (0/1 JAK2V617F-positive), peripheral arterial thrombosis (1/2 JAKV617F-positive), or hydroxyurea, interferon or anagrelide use. When controlled for gender, there were no significant differences in leukocyte count, hemoglobin concentration, reticulocyte count or platelet count between JAK2V617F-positive and -negative individuals. JAK2V617F status did not correlate with a family history of an MPD, present in 6 JAK2V617F-negative patients (3 male, 3 female) and 9 JAK2V617F-positive patients (2 male, 7 female). During the study period, phenotypic conversion occurred in 5 patients with a median disease duration of 10 years (range 3–20 years); 3 converted to polycythemia vera (2/3 JAK2V617F positive) and 2 to idiopathic myelofibrosis (both JAK2V617F-positive). In those patients with phenotypic conversion (3 men and 2 women), the JAK2V617F allele percentages either increased to or were at the upper end of the cohort JAK2V617F allele percentage distribution. In summary, in a well-defined, prospectively studied ET patient population, the neutrophil JAK2V617F allele burden was higher in men than women and in some women, JAK2V617F expression was restricted to platelets alone. While phenotypic conversion was associated with an increasing or high JAK2V617F allele percentage, neutrophil JAK2V617F did not correlate with platelet-mediated micro- or macrovascular events nor was there any correlation with hemoglobin, white cell count or platelet count. We conclude that JAK2V617F allele burden in ET is gender-specific and that comparison of JAK2V617F-positive and -negative ET patients must consider both neutrophil and platelet quantitative JAK2V617F allele expression.

Evidence for a role for Gαi1 in mediating weak agonist-induced platelet aggregation in human platelets: reduced Gαi1 expression and defective Gi signaling in the platelets of a patient with a chronic bleeding disorder

AbstractWe have examined platelet functional responses and characterized a novel signaling defect in the platelets of a patient suffering from a chronic bleeding disorder. Platelet aggregation responses stimulated by weak agonists such as adenosine diphosphate (ADP) and adrenaline were severely impaired. In comparison, both aggregation and dense granule secretion were normal following activation with high doses of collagen, thrombin, or phorbol-12 myristate-13 acetate (PMA). ADP, thrombin, or thromboxane A2 (TxA2) signaling through their respective Gq-coupled receptors was normal as assessed by measuring either mobilization of intracellular calcium, diacylglycerol (DAG) generation, or pleckstrin phosphorylation. In comparison, Gi-mediated signaling induced by either thrombin, ADP, or adrenaline, examined by suppression of forskolin-stimulated rise in cyclic AMP (cAMP) was impaired, indicating dysfunctional Gαi signaling. Immunoblot analysis of platelet membranes with specific antiserum against different Gα subunits indicated normal levels of Gαi2,Gαi3,Gαz, and Gαq in patient platelets. However, the Gαi1level was reduced to 25% of that found in normal platelets. Analysis of platelet cDNA and gDNA revealed no abnormality in either the Gαi1 or Gαi2 gene sequences. Our studies implicate the minor expressed Gαi subtype Gαi1 as having an important role in regulating signaling pathways associated with the activation of αIIbβ3 and subsequent platelet aggregation by weak agonists.

Evidence for two alternatively spliced forms of phospholipase C-beta2 in haematopoietic cells.

Alternatively spliced forms have been reported for several phospholipase C (PLC) isozymes, but not for PLC-beta2, the most abundant PLC-beta in platelets. PLC-beta2 cDNA cloned from the HL-60-cell cDNA library is 3543 bases long, coding for 1181 amino acids. Compared with the published sequence, a deletion of 45 nucleotides (2755-2799 nt, amino acids 864-878) was detected in platelet and leucocyte mRNA amplified by reverse transcription (RT) polymerase chain reaction (PCR) using primers corresponding to 1814-1838 nt (forward) and 3328-3352 nt (reverse). Amplification of genomic DNA using primers corresponding to 2575-2596 nt and 2864-2885 nt yielded a approximately 750 bp product; restriction analysis and sequencing revealed the 45-bp exon flanked by introns of 198 bp and 118 bp. Amplification of leucocyte and platelet cDNA using the same primers yielded products of approximately 310 nt and approximately 265 nt, with (PLC-beta2a) and without (PLC-beta2b) the 45-nt sequence. Thus, two alternatively spliced forms (1181 and 1166 amino acids) of PLC-beta2 are generated in haematopoietic cells. They differ in the carboxyl terminal sequence implicated in interaction of PLC-beta enzymes with Galphaq, particulate association and nuclear localization. We propose that the PLC-beta2 splice variants may be regulated differentially with distinct roles in signal transduction.

DNA sequence of the canine platelet β3 gene from cDNA: Comparison of canine and mouse β3 to segments that encode alloantigenic sites and functional domains of β3 in human beings

The platelet glycoprotein complex αIIbβ3 is required for platelet-fibrinogen binding and platelet aggregation. This study was designed to characterize the nucleotide sequence of the canine platelet β3 gene from cDNA. The nucleotide and deduced amino acid sequences of the canine β3 gene were 92% and 96% homologous, respectively, with the sequences previously established for the β3 gene of human beings. Within the β3 gene, the nucleotide sequence of cDNA prepared from canine platelets shared homology of 89% for the cytoplasmic domain, 93% for the transmembrane domain, 92% for the extracellular domain, 94% for the arginine-glycineaspartic acid (RGD) binding domain, and 97% for the region associated with Ca2+-dependent stabilization of the αIIbβ3 fibrinogen-binding pocket. The deduced amino acid sequence of canine β3 was 100%, 97%, 96%, and 95% homologous with the cytoplasmic, transmembrane, extracellular, and RGD-binding domains, respectively, and was 100% homologous with the region associated with Ca2+-dependent stabilization of the αIIbβ3 fibrinogen-binding pocket of β3 in human beings. The canine platelet cDNA signal peptide segment of the β3 gene encodes for 22 amino acids, as compared with 26 amino acids previously reported for human beings. The deduced amino acid sequence of canine β3 corresponds to the high-frequency allelic form for five of the six alloantigenic sites reportedly associated with human platelets: Leu33Leu40Pro407Arg489Arg636. The apparent amino acid residue in position 143 (Pen alloantigen) of canine platelet β3 is histidine compared with arginine in human beings. Knowledge of the β3 gene nucleotide sequence of normal dogs will facilitate the understanding of platelet αIIbβ3 structure-function relationships.

Cloning and Expression of Canine Glycoprotein Ibα

The interaction of the glycoprotein (GP) Ib-IX-V complex with von Willebrand factor (vWF) is critical in initiation of haemostasis and thrombosis through platelet adhesion to damaged endothelium. The binding site for vWF resides within the GPIbalpha subunit of the complex. To further define the physiological function of platelet GPIbalpha we cloned and expressed the canine GPIbalpha cDNA. A canine platelet cDNA library was constructed and screened with a randomly primed 32P-labeled 1041-base-pair restriction fragment of the human GPIbalpha cDNA. Analysis of 23 clones demonstrated that the canine GPIbalpha cDNA is 2530 nucleotides in length and includes a short 5' untranslated segment of 42 nucleotides followed by a signal peptide of 16 amino acids, a mature peptide of 645 amino acids and a 3' noncoding region of 455 nucleotides. A single intron of 142 nucleotides, 6 nucleotides upstream from the ATG translation initiation codon was identified in the canine gene in a similar location to that present in the human gene. Chinese hamster ovary cells that stably express human GPIbbeta and GPIX were transfected with the canine GPIbalpha cDNA. Canine GPIbalpha was expressed on the surface of these cells and bound vWF in the presence of botrocetin. The binding of vWF was inhibited by an anti-vWF human monoclonal antibody known to inhibit vWF binding to GPIbalpha. The results of this investigation will allow the development of reagents to study the physiological function of GPIbalpha in an animal model.

Role of P2Y1 purinoceptor in ADP-induced platelet activation

ADP acts as an agonist of platelet aggregation via specific receptors which are still to be characterised. Amplification by PCR of a human platelet cDNA library confirmed the presence of mRNA of the P2Y1 receptor in platelets. In order to determine if these P2Y1 receptors were involved in ADP-induced platelet activation, we determined the effects of A3P5PS, an antagonist of the P2Y1 receptor, on the binding of [ 33P]2-MeS-ADP, a potent analogue of ADP. We found that A3P5PS displaced about 27% of [ 33P]2-MeS-ADP binding, a receptor population which has been shown to be resistant to treatment with clopidogrel, a selective anti-ADP agent. A3P5PS specifically inhibited 2-MeS-ADP-induced shape change and calcium increase but did not affect adenylyl cyclase down-regulation. 2-MeS-ADP-induced platelet aggregation was also inhibited by A3P5PS but was restored when platelets were further activated by serotonin, a non-aggregating compound, therefore suggesting that P2Y1-mediated stimulation is an absolute prerequisite for ADP to induce platelet aggregation and a key event for platelet activation and aggregation to occur. These results therefore show that ADP-induced aggregation cannot be attributed to activation of P2Y1 alone, but must be attributed to the simultaneous activation of the high affinity receptor (P2Y1) and a low affinity receptor of ADP (still to be discovered), each of them essential, but neither able to trigger aggregation alone.

Molecular Basis for ADP-induced Platelet Activation: II. THE P2Y1 RECEPTOR MEDIATES ADP-INDUCED INTRACELLULAR CALCIUM MOBILIZATION AND SHAPE CHANGE IN PLATELETS

ADP is an important platelet agonist causing shape change from smooth discoid shape to spiculated spheres and platelet aggregation. However, the molecular mechanisms involved in ADP-induced platelet activation have not been elucidated. We demonstrated earlier the existence of two distinct ADP receptors on platelets, one coupled to phospholipase C, P2TPLC, and the other to inhibition of adenylyl cyclase, P2TAC (Daniel, J. L., Dangelmaier, C., Jin, J., Ashby, B., Smith, J. B., and Kunapuli, S. P. (1998) J. Biol. Chem. 273, 2024-2029), in addition to the previously described P2X1 receptor. Here we report the cloning of a cDNA clone encoding the P2Y1 receptor from a human platelet cDNA library by homology screening with radiolabeled P2Y1-P2Y6 receptor cDNAs. ADP or 2-methyl(thio)-ADP-induced intracellular calcium increases were inhibited by the P2Y1 receptor-specific antagonists, adenosine 3'-phosphate 5'-phosphosulfate (A3P5PS), adenosine 3'-phosphate 5'-phosphate (A3P5P), and adenosine 2'-phosphate 5'-phosphate (A2P5P), in a concentration-dependent manner, but not by ARL 66096 or alpha, beta-MeATP. A3P5PS, A3P5P, and A2P5P also inhibited the shape change of aspirinated platelets induced by 10 microM ADP or 3 microM 2-methyl-(thio)-ADP in a concentration-dependent manner, with complete inhibition occurring at 300 microM. On the other hand ARL 66096 (100 nM), a potent P2TAC antagonist and alpha, beta-methylene-ATP (40 microM), a P2X1 receptor agonist, had no effect on ADP-induced platelet shape change. On the contrary, ADP-induced inhibition of adenylyl cyclase was blocked by ARL 66096, but not by alpha, beta-MeATP or the P2Y1 receptor-specific antagonists, A3P5PS, A3P5P, or A2P5P. These results demonstrate the role of the P2Y1 receptor in ADP-induced platelet shape change and calcium mobilization and support the idea that several P2 receptors are involved in the regulation of different aspects of platelet stimulus-response coupling.

The Genetic Defect in Two Well-Studied Cases of Bernard-Soulier Syndrome: A Point Mutation in the Fifth Leucine-Rich Repeat of Platelet Glycoprotein Iba

Bernard-Soulier syndrome (B-Ss) is a rare congenital bleeding disorder caused by abnormal giant platelets, thrombocytopenia, and defective glycoprotein (GP) Ib-V-IX, the adhesion receptor for von Willebrand factor (vWF). This report describes the molecular defect in two related individuals with well-established B-Ss whose platelets exhibit decreased GPIb-IX and normal GPV on their surfaces. The GPIb-V-IX genes of the two patients were analyzed by Southern blotting, hetero-duplex analysis, and polymerase chain reaction (PCR) amplification/sequencing. A point mutation was found in codon 129 of the GPIb alpha gene that results in the substitution of proline for leucine in the first position of the fifth leucine-rich glycoprotein repeat of the mature gene product. The mutation (CTC: leucine, wild-type to CCC: proline, mutant) eliminates a Sac I restriction site, facilitating analysis of the mutation in the propositi (both homozygotes), unaffected family members (8 heterozygotes and 8 wild-type), and 58 normal controls (116 wild-type alleles). The status of the genomes was confirmed by the sequencing of platelet cDNA. The mutation does not affect transcription of the Ib-IX genes, as estimated by PCR and Northern blot analysis, but it does inhibit surface expression of the receptor as assessed by transient transfection of mutant and wild-type GPIb alpha genes into mouse Ib beta-IX L cells. Many of the cells (43%) transfected with the normal gene express surface GPIb alpha, whereas untransfected cells and those transfected with the mutant gene lack surface GPIb alpha entirely. Patient platelets were tested both for vWF binding in the presence of ristocetin and for surface GPIb-IX expression. In these instances, despite their inability to agglutinate with ristocetin and vWF, patient platelets exhibit about 40% of normal vWF binding and 40% of normal Ib-IX surface antigens. The results suggest that the described mutation (GPIb alpha: Leu129 -> Pro) affects the conformation of the GPIb-V-IX receptor, alters its availability on platelet surfaces, and causes the observed Bernard-Soulier phenotype.

Complementary DNA cloning of the alternatively expressed endothelial cell glycoprotein Ib beta (GPIb beta) and localization of the GPIb beta gene to chromosome 22.

Glycoprotein Ib beta (GPIb beta) exists in platelets disulfide-linked to glycoprotein Ib alpha (GPIb alpha), a major receptor for von Willebrand factor. Both GPIb alpha and GPIb beta are expressed in endothelial cells (EC). While the GPIb alpha mRNA and protein appear similar in platelets and EC, EC GPIb beta mRNA is larger than platelet GPIb beta and encodes a larger protein. We have cloned and sequenced EC GPIb beta cDNA and report a 2793-nucleotide sequence which contains a 411-amino acid open reading frame. The EC sequence contains all of the platelet cDNA sequence and all but three amino acids of the primary translation product. Like the genes encoding GPIb alpha, GPIX, and GPV, the GPIb beta gene appears simple in structure. Using human hamster hybrids, we have localized the GPIb beta gene to chromosome 22pter-->22q11.2. When we examined poly (A)+ RNA from several human tissues for GPIb beta mRNA expression, we found that GPIb beta mRNA was expressed in a variety of tissues but was most abundant in heart and brain, while GPIb alpha and GPIX mRNA expression was found only in lung and placenta at very low levels. The broad distribution of GPIb beta mRNA suggests that it may be playing a role different than or additional to its function in platelets.

Human platelet glycoprotein V: characterization of the polypeptide and the related Ib-V-IX receptor system of adhesive, leucine-rich glycoproteins.

Human platelet glycoprotein (GP) V (M(r) 83,300), whose primary structure is reported here, is a part of the Ib-V-IX system of surface glycoproteins (GPs Ib alpha, Ib beta, V, IX) that constitute the receptor for von Willebrand factor (vWf) and mediate the adhesion of platelets to injured vascular surfaces in the arterial circulation, a critical initiating event in hemostasis. System members share physical associations, leucine-rich glycoprotein (LRG) structures, and a congenital deficiency state, Bernard-Soulier syndrome. With PCR techniques and platelet cDNA templates, 1.4 kb of GP V cDNA sequence was obtained that encodes 469 GP V amino acids. A genomic 3.5-kb BamHI fragment was then isolated that includes 3.46 kb of GP V cDNA sequence: the 1.7-kb open reading frame plus 2 bases of the 5' and 1.8 kb of the 3' untranslated regions. Northern blot analysis reveals three GP V platelet transcripts of 3.8, 4.2, and 5.2 kb. A 16-amino acid signal peptide is present. Mature GP V is a 544-amino acid transmembrane protein with a 504-amino acid extracellular domain that encompasses a set of 15 tandem LRG repeats in a "flank-LRG center-flank" array [Roth, G. J. (1991) Blood 77, 5-19] along with eight putative N-linked glycosylation sites and cleavage sites for thrombin and calpain. GP V is a transmembrane, adhesive LRG protein that plays an undefined, but potentially critical, role in the expression and/or function of the Ib-V-IX receptor for vWf/shear-dependent platelet adhesion in arteries.

Partial purification of a GTPase-activating protein for rap2b from bovine brain membranes

Rap2n is a ras-related GTP-binding protein isolated from a human platelet cDNA library. It shares 90% similarity to the previously described rap2a and is closely related to rap1a (Krev-1, smgp21), which has been shown to possess reversion of transformation activity in Kirsten ras transformed 3T3 cells. In this study we have partially purified a protein from bovine brain membranes which stimulates the GTPase activity of rap2b. This rap2b GTPase-activating protein (GAP) activity is not immunoreactive with antibodies specific for rap1 GAP or ras GAP, yet displays limited GTPase stimulatory activity toward rap1. This result differs from the previously described rap1 GAP which is highly specific for rap1. When the rap2 GAP activity is analyzed by coomassie staining, an enrichment of a ≈ 55 kDa protein is observed providing further evidence of a distinct rap2 GAP.

Isolation and sequence of a cDNA encoding human platelet phosphofructokinase

A cDNA encoding human platelet 6-phosphofructokinase (PFK; EC 2.7.1.11) has been isolated from a human lymphocyte Raji cell line cDNA library using a cDNA for human muscle PFK as a probe. The platelet cDNA contains 900bp of carboxy terminal coding sequence and 238bp of downstream untranslated region. The deduced amino acid sequence shows 71% identity to the amino acid sequence for the human muscle isoenzyme and 63% identity to the human liver isoenzyme. Almost all of the amino acid residues contributing to catalytic and effector sites in the three isoenzymes are conserved. The platelet gene has been assigned to chromosome 10p15.2-p15.3 by using the cDNA clone as a biotinylated probe against human chromosome spreads (Morrison et al. 1991, submitted to Human Genetics).

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.

A 13-mer Peptide Straddling the Leucine33/Proline33 Polymorphism in Glycoprotein IIIa Does Not Define the PLA1 Epitope

Abstract 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.

Cloning of the cDNA for human 12-lipoxygenase.

A full-length cDNA clone encoding 12-lipoxygenase (arachidonate:oxygen 12-oxidoreductase, EC 1.13.11.31) was isolated from a human platelet cDNA library by using a cDNA for human reticulocyte 15-lipoxygenase as probe for the initial screening. The cDNA had an open reading frame encoding 662 amino acid residues with a calculated molecular weight of 75,590. Three independent clones revealed minor heterogeneities in their DNA sequences. Thus, in three positions of the deduced amino acid sequence, there is a choice between two different amino acids. The deduced sequence from the clone plT3 showed 65% identity with human reticulocyte 15-lipoxygenase and 42% identity with human leukocyte 5-lipoxygenase. The 12-lipoxygenase cDNA recognized a 3.0-kilobase mRNA species in platelets and human erythroleukemia cells (HEL cells). Phorbol 12-tetradecanoyl 13-acetate induced megakaryocytic differentiation of HEL cells and 12-lipoxygenase activity and increased mRNA for 12-lipoxygenase. The identity of the cloned 12-lipoxygenase was assured by expression in a mammalian cell line (COS cells). Human platelet 12-lipoxygenase has been difficult to purify to homogeneity. The cloning of this cDNA will increase the possibilities to elucidate the structure and function of this enzyme.

RAP2B: a RAS-related GTP-binding protein from platelets.

A platelet cDNA expression library was screened with the monoclonal antibody M90, which recognizes a specific epitope on RAS-encoded p21 proteins (amino acids 107-130). DNA sequence analysis of one clone revealed that it encoded a partial amino acid sequence of a protein closely related to RAP2, which we have named RAP2B. A repeated screening of the platelet cDNA library with an internal Ava I fragment of the RAP2B cDNA allowed the isolation of a full-length cDNA for the RAP2B sequence. RAP2B is 90% identical to RAP2 at the amino acid level with the most variability at the carboxyl terminus of the protein. Oligonucleotides were synthesized to complete the amino acid sequence of the RAP2B protein and the entire sequence was expressed in Escherichia coli. Analysis of crude soluble extracts indicated that RAP2B was a Mr 22,000 protein that specifically bound GTP on blots. Moreover, incubation of similar extracts with the catalytic subunit of cAMP-dependent protein kinase did not cause phosphorylation of RAP2B, as had been observed for the closely homologous proteins, RAP1A and RAP1B. These results suggest that RAP2B, like the other RAP proteins, is a low molecular weight GTP-binding protein in human platelets.

Circulating human blood platelets retain appreciable amounts of poly (A) + RNA

Platelets lack a nucleus and are usually considered to be incapable of protein synthesis due to an apparent lack of messenger RNA, precluding the construction of platelet cDNA libraries and hindering the cloning of authentic platelet cDNA's. We reasoned that vestigial amounts of messenger RNA may remain in platelets when they first separate from the megakaryocyte and circulate in the peripheral blood. We isolated poly (A) + RNA from platelets obtained by pheresis of individuals with elevated blood platelet counts due to a myeloproliferative syndrome termed essential thrombocythemia. Northern blots using probes for platelet glycoprotein Ib indicate that the poly (A) + RNA obtained from the platelets of these donors is, in fact, derived from platelets. Cell free translation studies using the platelet poly (A) + RNA indicate that the material is translationally active. We conclude that, contrary to prevailing information, circulating human blood platelets retain appreciable amounts of poly (A) + RNA and that this RNA can be harvested by the described approach. The poly (A) + RNA provides templates for the synthesis of cDNA's that code for platelet proteins.