Increased phosphorylations of proteins involved in the expression of the physiologic response of platelets in SHR rats

In primary hypertension, phospholipase C (PLC) is hypersensitive in several target tissues (platelets, vascular smooth muscle cells, aortic fibroblasts). Protein kinase C (PKC) and myosin light chain kinase (MLCK), which are physiologically activated by PLC-triggered second messengers (diacylglycerol and Ca2+ ions, respectively), phosphorylate specific proteins closely involved in the cell functional responses. In this study, we have examined and compared between platelets of spontaneously hypertensive rats (SHR) and their normotensive controls Wistar-Kyoto (WKY), the patterns of protein phosphorylation obtained either with the receptor-mediated agonist thrombin (i.e. which acts via PLC) or with direct activators of the protein kinases, PKC and MLCK. Activation by thrombin of 32P-prelabeled platelets induced incorporation of radioactivity into two proteins, P20 (myosin light chain) and P47. The curves obtained when platelets were challenged with either increasing doses of thrombin (0.025-0.3 U/ml) for 20 sec or with a low dose of the agent (0.1 U/ml) for up to 1 min, revealed that phosphorylation of the target proteins of PKC (P47) and of MLCK (P20) were significantly enhanced in platelets of SHR compared to WKY. In contrast, direct activation of PKC by phorbol ester and of MLCK by the calcium ionophore A23187 evoked the selective phosphorylation of the respective target proteins, P47 and P20, to a similar extent in platelets of SHR and WKY. Taken together, these results demonstrate that a physiological agonist (thrombin) induces an enhanced phosphorylation of intracellular proteins in platelets of SHR.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specific binding of [alpha-32P]GTP to cytosolic and membrane-bound proteins of human platelets correlates with the activation of phospholipase C.

We have assessed the binding of [alpha-32P]GTP to platelet proteins from cytosolic and membrane fractions. Proteins were separated by NaDodSO4/PAGE and electrophoretically transferred to nitrocellulose. Incubation of the nitrocellulose blots with [alpha-32P]GTP indicated the presence of specific and distinct GTP-binding proteins in cytosol and membranes. Binding was prevented by 10-100 nM GTP and by 100 nM guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]) or GDP; binding was unaffected by 1 nM-1 microM ATP. One main GTP-binding protein (29.5 kDa) was detected in the membrane fraction, while three others (29, 27, and 21 kDa) were detected in the soluble fraction. Two cytosolic GTP-binding proteins (29 and 27 kDa) were degraded by trypsin; another cytosolic protein (21 kDa) and the membrane-bound protein (29.5 kDa) were resistant to the action of trypsin. Treatment of intact platelets with trypsin or thrombin, followed by lysis and fractionation, did not affect the binding of [alpha-32P]GTP to the membrane-bound protein. GTP[gamma S] still stimulated phospholipase C in permeabilized platelets already preincubated with trypsin. This suggests that trypsin-resistant GTP-binding proteins might regulate phospholipase C stimulated by GTP[gamma S].     

Identification of the pertussis toxin-sensitive G proteins in platelets, megakaryocytes, and human erythroleukemia cells

Guanine nucleotide-binding regulatory proteins, or G proteins, mediate the interaction of agonist receptors on the platelet surface with phospholipase C and adenylyl cyclase. To better understand this process, we have used several approaches to identify which G proteins are present in platelets, normal human megakaryocytes, and human erythroleukemia (HEL) cells, a leukemic cell line with megakaryocytic features. Because platelet and HEL cell responses to thrombin are inhibited by pertussis toxin, we have focused upon the members of the Gi family, whose alpha subunits can be ADP-ribosylated by that toxin. Western blots with antisera specific for Gi alpha demonstrated the presence in both platelets and HEL cells of the three best-described forms of this protein: Gi alpha 1, Gi alpha 2, and Gi alpha 3. Based upon immunoprecipitation studies with [35S]-methionine-labeled HEL cells, their relative abundance appears to be Gi alpha 2 much greater than Gi alpha 3 greater than Gi alpha 1. A HEL cell cDNA library screened with the Gi alpha antisera produced clones encoding Gi alpha 2 and Gi alpha 3 that had sequences similar to those reported from other sources. Gi alpha-specific probes created from these cDNA clones confirmed the presence of mRNA encoding Gi alpha 2 and Gi alpha 3 in both platelets (by Northern blotting) and megakaryocytes (by in situ hybridization). Thus the pertussis toxin substrates that have previously been detected in platelets and HEL cells are shown to be members of the Gi alpha family, all of which are candidates for interaction with receptors for thrombin and other agonists.     

Identification of Gz alpha as a pertussis toxin-insensitive G protein in human platelets and megakaryocytes

G proteins mediate the interaction between cell surface receptors and intracellular effectors. Recent studies have shown that human retina and rat brain contain mRNA encoding a novel 40-Kd G protein alpha subunit referred to as Gz alpha. Studies with an antiserum selective for the predicted sequence of this protein have suggested that a similar protein is present in human platelets and is phosphorylated during platelet activation. To better understand the structure and function of this protein, the present studies examine its sequence in platelets and compare its abundance in human platelets, megakaryocytes, and two megakaryoblastic cell lines, HEL cells and Dami cells. Three different Gz alpha-selective antisera reacted with a 40-Kd protein in platelet membranes. None of these detected a corresponding protein in HEL or Dami cells, despite the presence in both cell lines of proteins recognized by antisera selective for three members of the Gi alpha family. Northern blotting with a Gz alpha-specific probe prepared from retinal Gz alpha showed two hybridizing species in platelet RNA: a major band at 3.5 kb and a minor band at 2.2 kb. Both were detectable in HEL and Dami cells, but at greatly reduced levels compared with platelets. RNA encoding Gz alpha was also detected in individual human megakaryocytes by in situ hybridization. The amount present approached that of Gi alpha 2' the most abundant of the Gi alpha species present in platelets. The complete sequence of the platelet homolog to Gz alpha was determined from platelet RNA amplified by the polymerase chain reaction. The encoded protein was the same as those obtained in brain and retina. Thus, based on immunoreactivity and nucleotide sequencing, platelets and megakaryocytes contain substantial quantities of a protein identical to brain and retinal Gz alpha. The paucity of Gz alpha protein and RNA in the megakaryoblastic cell lines suggests that either there has been a selective loss of the ability to synthesize Gz alpha from these cells or that Gz alpha appears at a later stage in megakaryocyte development than does Gi alpha.     

Membrane glycoprotein IV (CD36) is physically associated with the Fyn, Lyn, and Yes protein-tyrosine kinases in human platelets.

Activation of platelets with thrombin and other agonists causes a rapid increase in the phosphorylation of multiple proteins on tyrosine. To identify candidate protein-tyrosine kinases (PTKs; EC 2.7.1.112) that may be responsible for these phosphorylation events, we analyzed the expression of seven Src-family PTKs and examined the association of these kinases with known platelet membrane glycoproteins. Five Src-related PTKs were detected in platelets: pp60SRC, pp60FYN, pp62YES, pp61HCK, and two LYN products of Mr 54,000 and 58,000. The Fgr and Lck PTKs were not detected. Although strict comparative quantification of protein levels was not possible, pp60SRC was detected at higher levels than any of the other kinases. In addition, glycoprotein IV (GPIV, CD36), one of the major platelet membrane glycoproteins, was associated in a complex with the Fyn, Yes, and Lyn proteins in platelet lysates. Similar complexes were also found in two GPIV-expressing cell lines, C32 melanoma cells and HEL cells. Since PTKs appear to be involved in stimulus-response coupling at the plasma membrane, these results suggest that ligand interaction with GPIV may activate signaling pathways that are triggered by tyrosine phosphorylation.     

Protein phosphorylation and intracellular free calcium in platelets of patients with essential hypertension.

Platelet intracellular free calcium concentration [Ca2+]i from patients with essential hypertension has been found to be elevated, but the intracellular effects of this increase are still unclear. As protein phosphorylation is an important regulatory step in cell activation and increased protein phosphorylation has been demonstrated in platelets from hypertensive animals, we investigated protein phosphorylation and [Ca2+]i in platelets from patients with essential hypertension and age-matched normotensives. We measured the 32P incorporation into a 20 kDa protein and a 47 kDa protein in 17 hypertensive patients and 20 normotensive, age-matched subjects. The [Ca2+]i was measured with the fluorescent dye fura-2. Protein phosphorylation and [Ca2+]i were assessed in unstimulated platelets and after exposure of the cells to 0.1 and 0.25 U/mL thrombin at 20, 60, and 300 sec. In addition we assessed the activity of protein kinase C by incubating the platelets with phorbol-ester TPA at 20, 60, and 300 sec. Basal phosphorylation of the two proteins was not different between the two groups. After exposure of the platelets to thrombin 32P, incorporation into the 20 kDa protein and the 47 kDa protein was significantly increased in platelets from hypertensive patients at all times. Furthermore, the specific stimulation of protein kinase C with TPA resulted in a significantly higher phosphorylation of the 47 kDa protein, whereas the 20 kDa protein was not phosphorylated after incubation with TPA for 1 min. Basal [Ca2+]i was higher in platelets from hypertensive patients (124 +/- 7 nmol/L v 104 +/- 5 nmol/L, P less than .05), although there was a wide overlap between the two groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization and regulation of the receptor tyrosine kinase Tie-1 in platelets

The receptor tyrosine kinase Tie-1 is expressed predominantly on endothelial cells where it has an essential role in blood vessel formation. Targeted disruption of the Tie-1 gene results in a lethal phenotype with severe disruption to the normal integrity of the vasculature. In an examination of Tie-1 in vivo, we observed a significant pool of the receptor present in the circulation associated with the platelet fraction. Western blotting reveals the platelet form of Tie-1 to be a protein of approximately 110 kDa, this contrasts with the 135/125-kDa doublet found in endothelial cells. Platelet activation results in increased surface expression of Tie-1. The closely related receptor tyrosine kinase Tie-2/Tek is not present in platelets. Endothelial Tie-1 undergoes metalloprotease-mediated ectodomain cleavage in response to phorbol ester and other agonists. Tie-1 cleavage leads to release of the extracellular domain and generation of a cell-associated intracellular domain with signalling capacity. The potential for cleavage was investigated in platelets. In contrast to endothelial Tie-1, phorbol ester does not stimulate truncation of the platelet receptor, suggesting these cells lack one or more components of the regulated metalloprotease system controlling Tie-1. These data demonstrate the Tie-1 receptor tyrosine kinase is present on platelets and its surface expression is regulated. Furthermore, platelet Tie-1 differs significantly from the endothelial receptor. Platelet Tie-1 has the potential to modulate endothelial function by competing for any Tie ligands and may have signalling roles important in controlling aspects of platelet behaviour.     

Activation of protein kinase C in platelets by epinephrine and A23187: correlation with fibrinogen binding

Activation of protein kinase C (PKC), as revealed by phosphorylation of a 47 kd protein (p47), occurs in platelets stimulated by some agonists (eg, thrombin or phorbol esters). It is not known if activation of PKC occurs with pairs of agonists, such as epinephrine and A23187, that do not individually phosphorylate p47, nor is it known what role the concentration of cytoplasmic Ca++ ([Ca++]i) plays in these events. We stimulated aequorin-loaded platelets with subaggregating concentrations of epinephrine and A23187, neither of which by itself phosphorylated p47. The combination of agonists resulted in p47 phosphorylation, an increase in platelet-bound fibrinogen, and aggregation, but only if the concentration of each agonist was sufficient to increase [Ca++]i if it was added separately. Subaggregating concentrations of A23187 alone released platelet fibrinogen and increased platelet membrane binding of [3H]-phorbol dibutyrate, but these were not enhanced by epinephrine. Epinephrine and A23187 did not increase production of diacylglycerol. Thus, epinephrine and A23187 together activate PKC by a mechanism that does not require phospholipase C or enhanced binding of PKC to the plasma membrane; PKC activation in turn is correlated with enhanced platelet fibrinogen binding and aggregation. These events require an initial elevation of [Ca++]i above a threshold.     

Identification of minor metabolites of phospholipid signal molecules in [ 32 P]P i -labelled platelets

Incubation of blood platelets with 32 P-labelled inorganic phosphate for 60 min leads to incorporation of radioisotope mainly into phosphatidylinositol (PI), phosphatidylinositol-4-phosphate (PIP) and phosphatidyl-4,5-bisphosphate (PIP 2 ) in resting platelets and into phosphatidic acid (PA) in activated platelets. Small amounts of other important phosphoinositide isomers also become labelled following platelet activation, among them the 3-phosphorylated derivatives. In addition, several other faintly labelled spots are visible on TLC separations. Three of these lipids have now been identified as lysophosphatidylinositol (lysoPI), lysophosphatidic acid (lysoPA) and CDP-diacylglcerol (CDP-DAG).[ 32 P]LysoPI was present in resting and activated platelets, whereas [ 32 P]lysoPA and [ 32 P]CDP-DAG were observed only upon platelet activation. The phosphoinositide cycle turns over without accumulation of [ 32 P]PA and [ 32 P]CDP-DAG in resting platelets. A large increase (as much as 40-fold) in the steady-state level of [ 32 P]PA is seen in thrombin-activated platelets. A slight increase in the steady-state levels of [ 32 P]CDP-DAG is accompanied by a similar increase in [ 32 P]PI and larger increases in [ 32 P]PIP and [ 32 P]PIP 2 (about 50%), which is indicative of a general increase in flux in the PPI cycle. Elevation of CDP-DAG levels is probably only a reflection of increased flux, whereas lysoPA and lysoPI have been reported to have diverse signalling functions in various cells.     

BCR-ABL oncoprotein is expressed by platelets from CML patients and associated with a special pattern of CrkL phosphorylation

Constitutive tyrosine phosphorylation of CrkL was recently demonstrated in platelets from chronic myelogenous leukaemia (CML) patients but BCR-ABL tyrosine kinase could not be detected in the platelet lysates. We studied platelets from 14 CML patients with different types of BCR-ABL mRNA and with maximal platelet counts ranging from 149 to 3069 × 10⁹/l. P2l0^BCR-ABL protein was detected by Western blotting in platelet lysates of 12/13 CML patients with active disease but not in the lysate of platelets from a Ph-positive acute lymphoblastic leukaemia (ALL) patient in remission or eight BCR-ABL-negative controls including one essential thrombocythaemia (ET) patient. Immunoblotting of p2l0^BCR-ABL-positive platelets lysates with anti-CrkL antibody revealed a CrkL triplet consisting of one unphosphorylated and two phosphorylated forms of the protein. This CrkL phosphorylation pattern was not observed in normal platelets or CML platelets treated with ABL tyrosine kinase inhibitor CGP57148B. The presence of BCR-ABL provides an explanation for the constitutive tyrosine phosphorylation of CrkL in CML platelets. As no correlation was observed between platelet counts and platelet BCR-ABL protein expression, thrombocytosis or thrombocythaemia in CML cannot be explained by constitutive BCR-ABL-mediated CrkL tyrosine phosphorylation.     

Identification of minor metabolites of phospholipid signal molecules in [(32)P]P(i)-labelled platelets

Incubation of blood platelets with (32)P-labelled inorganic phosphate for 60 min leads to incorporation of radioisotope mainly into phosphatidylinositol (PI), phosphatidylinositol-4-phosphate (PIP) and phosphatidyl-4,5-bisphosphate (PIP(2)) in resting platelets and into phosphatidic acid (PA) in activated platelets. Small amounts of other important phosphoinositide isomers also become labelled following platelet activation, among them the 3-phosphorylated derivatives. In addition, several other faintly labelled spots are visible on TLC separations. Three of these lipids have now been identified as lysophosphatidylinositol (lysoPI), lysophosphatidic acid (lysoPA) and CDP-diacylglcerol (CDP-DAG).[(32)P]LysoPI was present in resting and activated platelets, whereas [(32)P]lysoPA and [(32)P]CDP-DAG were observed only upon platelet activation. The phosphoinositide cycle turns over without accumulation of [(32)P]PA and [(32)P]CDP-DAG in resting platelets. A large increase (as much as 40-fold) in the steady-state level of [(32)P]PA is seen in thrombin-activated platelets. A slight increase in the steady-state levels of [(32)P]CDP-DAG is accompanied by a similar increase in [(32)P]PI and larger increases in [(32)P]PIP and [(32)P]PIP(2) (about 50%), which is indicative of a general increase in flux in the PPI cycle. Elevation of CDP-DAG levels is probably only a reflection of increased flux, whereas lysoPA and lysoPI have been reported to have diverse signalling functions in various cells.     

Protein kinase-C-induced down-regulation of calcitonin receptors and calcitonin-activated adenylate cyclase in T47D and BEN cells

T47D human breast cancer cells and BEN human lung cancer cells were preincubated with the tumor-promoting phorbol ester phorbol 12-myristate 13-acetate (PMA). In both cell lines there was a decrease in the binding of 125I-labeled salmon calcitonin ([125I]sCT) which was dependent on the dose and time of exposure to PMA. The effect on binding comprised at least two components: the apparent affinity for binding of [125I]sCT was decreased by PMA, and the rate of internalization of bound [125I]sCT was increased more than 2-fold in the presence of PMA. By using dinitrophenol to inhibit cellular metabolic energy and, therefore, receptor internalization, the PMA effects on receptor affinity were dissociated from those on endocytosis. The effects on binding were reflected in a decreased stimulation by sCT of adenylate cyclase activity. This was specific for the calcitonin receptor system, since PMA had no effect on prostaglandin-E2-stimulated adenylate cyclase in the T47D cell. Protein kinase-C (PKC) was implicated in the inhibitory effects of PMA on both binding and adenylate cyclase activation, since inhibition was reduced by simultaneous incubation with the PKC inhibitors H7 and H8. These results suggest that PKC is capable of mediating down-regulation of the CT receptor, and this is most likely by phosphorylation of the receptor itself or an associated protein.     

Human platelets exert cytotoxic effects on tumor cells

Monocytes are thought to play a role in host resistance to tumor cell growth in animals and humans. In addition, platelets are known to be involved in tumor metastases. To investigate the interaction of these two cell types and their effect on tumor cells, human monocytes and platelets were examined using an in vitro monocyte-tumor cell cytotoxicity assay. Monocytes alone resulted in 32% +/- 1.5 (mean +/- SEM) tumor cell kill. When platelets were added to monocytes in a 1:1 ratio, an increase in cytotoxicity to 61% +/- 3.2 was observed. The cytotoxicity noted when platelets were added to a fixed number of monocytes and tumor cells was dependent on the number of platelets added. A decrease in cytotoxicity from 32% +/- 1.5 to 12% +/- 2.3 was observed when contaminating platelets were removed from monocyte preparations. Platelets added to tumor cells in the absence of any monocytes were also toxic, resulting in a maximum kill of 95% at a 4:1 platelet/tumor cell ratio. Secreted products of freshly isolated platelets may be responsible for much of the observed cytotoxicity, since supernatants from the platelets were toxic for tumor cells. Platelets pretreated with a cyclooxygenase inhibitor (ASA) or a lipoxygenase inhibitor had decreased cytotoxicity compared with untreated platelets. Our results indicate that products of platelet arachidonate metabolism are toxic for tumor cell lines. They also suggest that the role of the platelet must be considered when studying monocyte-tumor cell cytotoxicity.     

Adducin in platelets: activation-induced phosphorylation by PKC and proteolysis by calpain

Adducins are a family of cytoskeletal proteins encoded by 3 genes (alpha, beta, and gamma). Platelets express alpha and gamma adducins, in contrast to red blood cells that express alpha and beta adducins. During platelet activation with thrombin, calcium ionophore A23187, or phorbol 12-myristate 13-acetate, alpha and gamma adducins were phosphorylated by protein kinase C (PKC) as detected by an antibody specific for a phosphopeptide sequence in the highly conserved carboxy terminus. Platelet activation also led to adducin proteolysis; inhibition by calpeptin suggests that the protease was calpain. The kinase inhibitor staurosporine inhibited PKC phosphorylation of adducin and also inhibited proteolysis of adducin. Experiments with recombinant alpha adducin demonstrated that the PKC-phosphorylated form was proteolyzed at a significantly faster rate than the unphosphorylated form. The concentration of adducin in platelets was estimated at 6 microM, similar to the concentration of capping protein. Fractionation of platelets into high-speed supernatant (cytosol) and pellet (membrane and cytoskeleton) revealed a shift of PKC-phosphorylated adducin to the cytosol during platelet activation. Platelet aggregation detected turbidometrically was decreased in the presence of staurosporine and was completely inhibited by calpeptin. Thrombin-induced changes in morphology were assessed by confocal microscopy with fluorescein phalloidin and were not prevented by staurosporine or calpeptin. Our results suggest that regulation of adducin function by PKC and calpain may play a role in platelet aggregation.     

Crkl is constitutively tyrosine phosphorylated in platelets from chronic myelogenous leukemia patients and inducibly phosphorylated in normal platelets stimulated by thrombopoietin

Platelet functions such as aggregation and clot retraction are often abnormal in chronic mylogenous leukemia (CML) patients. However, the molecular mechanisms of these altered functions are unknown. As expression of the p210bcr-abl oncogene product, a constitutively active tyrosine kinase, is known to have an essential role in the pathogenesis of CML and tyrosine phosphorylation is intimately involved in various aspects of platelet activation, we examined the pattern of protein tyrosine phosphorylation in platelets from 15 CML patients by immunoblotting with a monoclonal antiphosphotyrosine antibody (4G10). Before and after stimulation with thrombin, the only consistent difference between normal and CML platelets was the presence of a tyrosine phosphorylated protein with a relative molecular weight of 39 kD. This tyrosine phosphorylated protein was identified as crid, an SH2, SH3 containing adapter protein. Thus, as previously demonstrated for neutrophils from CML patients, tyrosine phosphorylation of p39crkl persists in mature platelets. No tyrosine phosphorylation of crid was detected following stimulation with thrombin in normal platelets. However, crkl became incorporated into the Triton X-100 insoluble residue following thrombin stimulation in a manner dependent on platelet aggregation. Further, we found that crkl is an endogenous substrate for calpain, a protease that may be involved in postaggregation signaling processes. This suggests that crkl may be involved in the reorganization of the cytoskeleton during normal platelet aggregation and its tyrosine phosphorylation in CML platelets may contribute to the abnormal platelet function in CML patients. Finally, we found that thrombopoietin induces tyrosine phosphorylation of crk1 in normal platelets and FDCP cells genetically engineered to express human c-Mpl. This suggests that crk1 can be phosphorylated by a kinase other than p210bcr-abl and that crk1 may have a role in signaling by thrombopoietin.     

Phorbol esters sensitize platelets to activation by physiological agonists

Phorbol esters such as phorbol 12, 13-dibutyrate (PdBu; 40 to 200 nmol/L) or 12-O-tetradecanoyl phorbol 13-acetate (20 to 80 nmol/L) added to aspirinized platelet-rich plasma (PRP) 5 to 15 seconds prior to various platelet stimuli (epinephrine, ADP, prostaglandin endoperoxide analog U44069, collagen, PAF, or vasopressin) potentiate the rate and extent of aggregation and ATP secretion induced by those agonists. Platelet aggregation, but not secretion, is potentiated at low concentrations of agonists; platelet secretion is potentiated at higher concentrations of the platelet stimuli. Potentiation of platelet responses was also observed when the preincubation time with PdBu was extended to 12 minutes and also occurred in washed platelets. The potentiating effect of phorbol esters is not mediated by formation of arachidonate metabolites or by released ADP. The sensitizing effect of PdBu on platelet aggregation induced by epinephrine is unique, since in contrast to the other platelet stimuli it is also found at maximal concentrations of epinephrine and does not diminish with prolonged preincubation of platelets with PdBu. Activation of protein kinase C ranges from 20% to 80% over control after 1 to 10 minutes of platelet pretreatment with PdBu but dramatically increases after subsequent addition of a stimulus such as vasopressin. In contrast, agonist- induced myosin light chain phosphorylation is reduced after platelet pretreatment with PdBu. The results indicate that protein kinase C activation enhances platelet aggregation and dense granule secretion triggered by physiologic stimuli, although it desensitizes agonist- induced myosin light chain phosphorylation.     

The murine monoclonal antibody, 14A2.H1, identifies a novel platelet surface antigen

A murine monoclonal antibody 14A2.H1, raised against acute myeloid leukaemia cells, identifies a previously undescribed 27 kDa platelet surface glycoprotein which is expressed at low copy number (10(3)/platelet). MAb 14A2.H1 caused aggregation of platelets which was dependent on Fc gamma RII. Binding of the antibody to platelets was not altered by activation by thrombin or phorbol ester. In haemopoietic cell populations the antibody bound to megakaryocytes, monocytes (weakly), several myeloid leukaemic cell lines and fresh myeloid leukaemic blasts from some patients. Lymphocytes, lymphoid cell lines, neutrophils and haemopoietic progenitor cells were negative. Expression of the antigen was not restricted to haemopoietic cells as epithelial cells in tonsillar crypts and endothelial cells were positive.     

Glycoprotein IIIa is phosphorylated in intact human platelets

The glycoprotein IIb-IIIa complex (GP IIb-IIIa) is a multifunctional transmembrane protein on platelets. Its most completely described function is as a fibrinogen receptor that mediates platelet aggregation, but it is also involved in clot retraction, signal transduction, calcium transport, and other events. However, the mechanisms that regulate the functions of GP IIb-IIIa during platelet activation are largely unknown. One possible mechanism is phosphorylation, since several other receptors are regulated by this process. We found that GP IIIa, but not GP IIb, was phosphorylated in 32P-labeled platelets, predominantly on threonine residues. Furthermore, GP IIIa phosphorylation increased four-fold in platelets activated with thrombin or phorbol 12-myristate 13-acetate, but not at all in platelets treated with prostacyclin, an inhibitor of platelet activation. The thrombin-induced increase in phosphorylation was inhibited by pretreating platelets with prostacyclin or with staurosporin, a specific protein kinase C inhibitor. Thus, there is an increase in the level or turnover of phosphate on GP IIIa during platelet activation, most likely involving protein kinase C. This phosphorylation may regulate some aspect(s) of GP IIb-IIIa function.     

A ras-related protein is phosphorylated and translocated by agonists that increase cAMP levels in human platelets.

The antigenicity of platelet proteins was assayed against various monoclonal antibodies (mAbs) that recognize specific epitopes of the ras-encoded p21 protein. mAb M90, which detects the region of p21 protein within amino acids 107-130 and inhibits its GTP-binding activity, strongly reacted with a 22-kDa protein present in the particulate fraction of human platelets. Other mAbs against ras-encoded proteins, including Y13-259, which efficiently detects ras proteins from a variety of organisms, did not recognize the platelet 22-kDa protein. Transfer of the platelet 22-kDa protein to nitrocellulose paper showed that the protein binds [alpha-32P]GTP. Moreover, preincubation of the transferred protein with mAb M90 drastically reduced its GTP-binding activity. Treatment of platelets with iloprost, a prostacyclin analog, caused (i) a time-dependent increase of a 24-kDa protein that is recognized by mAb M90 in particulate and cytosolic fractions and (ii) the gradual decrease of the 22-kDa protein from the particulate fraction. When platelets were labeled with 32P and then treated with iloprost, the 24-kDa protein was found to be phosphorylated. The 32P-labeled 24-kDa protein was specifically immunoprecipitated by mAb M90. These results suggest that appearance of the 24-kDa protein results from phosphorylation of the 22-kDa protein, which shifts its mobility to a higher molecular mass area.     

Human platelets contain SNARE proteins and a Sec1p homologue that interacts with syntaxin 4 and is phosphorylated after thrombin activation: implications for platelet secretion

In response to thrombin and other extracellular activators, platelets secrete molecules from large intracellular vesicles (granules) to initiate thrombosis. Little is known about the molecular machinery responsible for vesicle docking and secretion in platelets and the linkage of that machinery to cell activation. We found that platelet membranes contain a full complement of interacting proteins-VAMP, SNAP-25, and syntaxin 4-that are necessary for vesicle docking and fusion with the plasma membrane. Platelets also contain an uncharacterized homologue of the Sec1p family that appears to regulate vesicle docking through its binding with a cognate syntaxin. This platelet Sec1 protein (PSP) bound to syntaxin 4 and thereby excluded the binding of SNAP-25 with syntaxin 4, an interaction critical to vesicle docking. As predicted by its sequence, PSP was detected predominantly in the platelet cytosol and was phosphorylated in vitro by protein kinase C (PKC), a secretion-linked kinase, incorporating 0.87 +/- 0.11 mol of PO4 per mole of protein. PSP was also specifically phosphorylated in permeabilized platelets after cellular stimulation by phorbol esters or thrombin and this phosphorylation was blocked by the PKC inhibitor Ro-31-8220. Phosphorylation by PKC in vitro inhibited PSP from binding to syntaxin 4. Taken together, these studies indicate that platelets, like neurons and other cells capable of regulated secretion, contain a unique complement of interacting vesicle docking proteins and PSP, a putative regulator of vesicle docking. The PKC-dependent phosphorylation of PSP in activated platelets and its inhibitory effects on syntaxin 4 binding provide a novel functional link that may be important in coupling the processes of cell activation, intracellular signaling, and secretion.