Pathogenetic analysis of three cases with a bleeding disorder characterized by defective platelet aggregation induced by Ca2+ ionophores

We report three cases of platelet dysfunction characterized by defective Ca2+ ionophore-induced platelet aggregation without impaired production of thromboxane A2 (TXA2). The patients had mild to moderate bleeding tendencies, and their platelet aggregation and secretion induced by ADP, collagen, arachidonic acid, stable TXA2 (STA2) and Ca2+ ionophore A23187 was defective or much reduced. However, ristocetin- or thrombin-induced platelet aggregation was normal. The analysis of second messenger formation showed that inositol 1,4,5-triphosphate formation or Ca2+ mobilization induced by thrombin, STA2 or A23187 was normal. Furthermore, the phosphorylation of 47 kDa protein (pleckstrin) and 20 kDa protein (myosin light chain, MLC) in response to those agonists was normal. These findings suggest that the defective site in the patients' platelets lies in the process distal to or independent of protein kinase C activation, Ca2+ mobilization and MLC phosphorylation.     

Mechanisms involved in the antiplatelet activity of magnesium in human platelets

In this study, magnesium sulphate dose-dependently (0.6-3.0 mmol/l) inhibited platelet aggregation in human platelets stimulated by agonists. Furthermore, magnesium sulphate (3.0 mmol/l) markedly interfered with the binding of fluorescein isothiocanate-triflavin to the glycoprotein (GP)IIb/IIIa complex in platelets stimulated by collagen. Magnesium sulphate (1.5 and 3.0 mmol/l) also inhibited phosphoinositide breakdown and intracellular Ca+2 mobilization in human platelets stimulated by collagen. Magnesium sulphate (3.0 mmol/l) significantly inhibited thromboxane A2 formation stimulated by collagen in platelets. Moreover, magnesium sulphate (1.5 and 3.0 mmol/l) obviously increased the fluorescence of platelet membranes tagged with diphenylhexatriene. In addition, magnesium sulphate (1.5 and 3.0 mmol/l) increased the formation of cyclic adenosine monophosphate (AMP) in platelets. Phosphorylation of a protein of Mr 47 000 (P47) was markedly inhibited by magnesium sulphate (1.5 mmol/l). In conclusion, the antiplatelet activity of magnesium sulphate may involve the following two pathways. (1) Magnesium sulphate may initially induce membrane fluidity changes with resulting interference of fibrinogen binding to the GPIIb/IIIa complex, followed by inhibition of phosphoinositide breakdown and thromboxane A2 formation, thereby leading to inhibition of both intracellular Ca2+ mobilization and phosphorylation of P47. (2) Magnesium sulphate might also trigger the formation of cyclic AM, ultimately resulting in inhibition of the phosphorylation of P47 and intracellular Ca+2 mobilization.     

Activation of human platelets by the rabbit anticardiolipin antibodies.

Affinity purified anticardiolipin antibodies (ACLA) raised in rabbits showed cross-reactivities with various negatively charged phospholipids as shown by both the solid phase enzyme-linked immunosorbent assay (ELISA) and inhibition studies. In ELISA, ACLA showed strong cross-reactivity to both sphingomyelin (SM) and phosphatidylethanolamine (PE), but the inhibition studies showed that ACLA failed to bind the aqueous suspensions of SM, PE, and PE/PC (1:1). ACLA bound to resting gel-filtered human platelets (GFP) as shown by both inhibition study and flow cytofluorometric analysis. Western blotting procedure showed that ACLA strongly cross-reacted to an 80-Kd plasma membrane protein. ACLA activated platelet response in a concentration-dependent manner. At less than 10 micrograms/mL, ACLA induced both platelet shape change to spiculate irregular forms as shown by scanning electron microscopy and the phosphorylation of 20-Kd protein. ACLA at more than 10 micrograms/mL caused platelet aggregation and secretion. The aggregation was inhibited by EDTA; aspirin; antimycin A plus 2-deoxyglucose; PGE1; and the F(ab')2 fragment of ACLA. It was not inhibited by monoclonal antibody to Fc receptor (MoAb FcR2). The biochemical events of ACLA-induced platelet response involved the elevation of (1) thromboxane A2 formation, (2) cytosolic free calcium ion concentration ([Ca2+]i), and (3) 47-Kd protein phosphorylation. In addition, the subaggregatory concentration of ACLA showed synergistic platelet activation with that concentration of thrombin, collagen, and epinephrine. The study showed the mechanism involved in ACLA-induced platelet responses.     

Inhibitory mechanisms of kinetin,a plant growth-promoting hormone,in platelet aggregation

Kinetin has been shown to have anti-aging effects on several different systems including plants and human cells. The aim of this study was to examine the detailed inhibitory mechanisms of kinetin in platelet aggregation. In this study, kinetin concentration-dependently (50-150 microM) inhibited platelet aggregation in human platelets stimulated by agonists. Kinetin (70 and 150 microM) also concentration-dependently inhibited intracellular Ca2+ mobilization and phosphoinositide breakdown in platelets stimulated by collagen (1 microg/ml). Kinetin (70 and 150 microM) significantly inhibited thromboxane A2 formation stimulated by collagen (1 microg/ml) and arachidonic acid (60 microM) in human platelets. In addition, kinetin (70 and 150 microM) significantly increased the formation of cyclic AMP. Intracellular pH values were measured spectrofluorometrically using the fluorescent probe BCECF-AM in platelets. The thrombin-evoked increase in pHi was markedly inhibited in the presence of kinetin (70 and 150 microM). Rapid phosphorylation of a platelet protein of molecular weight (Mr) 47000 (P47), a marker of protein kinase C activation, was triggered by collagen (1 microg/ml). This phosphorylation was inhibited by kinetin (70 and 150 microM). In conclusion, these results indicate that the anti-platelet activity of kinetin may be involved in the following pathways: kinetin's effects may initially be due to inhibition of the activation of phospholipase C and the Na+/H+ exchanger. This leads to lower intracellular Ca2+ mobilization, followed by inhibition of TxA2 formation and then increased cyclic AMP formation, followed by a further inhibition of the Na+/H+ exchanger, ultimately resulting in markedly decreased intracellular Ca2+ mobilization and phosphorylation of P47. These results suggest that kinetin has an effective anti-platelet effect and that it may be a potential therapeutic agent for arterial thrombosis.     

Evidence for a role for Galphai1 in mediating weak agonist-induced platelet aggregation in human platelets: reduced Galphai1 expression and defective Gi signaling in the platelets of a patient with a chronic bleeding disorder

We 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 Galphai signaling. Immunoblot analysis of platelet membranes with specific antiserum against different Galpha subunits indicated normal levels of Galphai2,Galphai3,Galphaz, and Galphaq in patient platelets. However, the Galphai1level was reduced to 25% of that found in normal platelets. Analysis of platelet cDNA and gDNA revealed no abnormality in either the Galphai1 or Galphai2 gene sequences. Our studies implicate the minor expressed Galphai subtype Galphai1 as having an important role in regulating signaling pathways associated with the activation of alphaIIbbeta3 and subsequent platelet aggregation by weak agonists.     

A defect of platelet release reaction in a patient with SLE: impaired platelet aggregation induced by phorbol ester with a normal phosphorylation of 40K protein

A 37-year-old female who suffered from SLE had a bleeding disorder. At the time of initial evaluation, the main disease demonstrated was a delta-storage pool deficiency. After this improved, a marked decrease of aggregation still remained, when induced by either ADP, epinephrine, collagen, A23187, thrombin, or PAF-acether. Although arachidonate-induced aggregation was slightly decreased, thromboxane B2 was produced normally in response to exogenous arachidonate. The patient's endoperoxides and/or thromboxane A2 aggregated aspirin-treated platelets, though her platelets were themselves unresponsive. Impaired aggregability induced by TPA (12-0-tetradecanoylphorbol-13-acetate) or OAG (1-oleoyl-2-acetyl-glycerol) was also found. However, the phosphorylation of P43 and P20 induced by several stimulators including CA++ ionophore was normal, using 32P-labelled platelets. It is suggested that TPA or OAG-induced platelet aggregation requires not only the phosphorylation of those proteins, but also another unknown mechanism after the phosphorylation, and that the platelet dysfunction of this patient was due to a defect of some mechanism involving Ca++ uptake or mobilization of cytoplasmic Ca++ from intracellular storage sites.     

A new congenital defect of platelet secretion: impaired responsiveness of the platelets to cytoplasmic free calcium

Summary . A 16-year-old boy with a bleeding disorder since infancy has a long bleeding time, normal platelet count and morphology and normal plasma factor-VIII activities. His platelets undergo normal shape change and primary aggregation in response to ADP but show defective 5-hydroxytryptamine (5-HT) secretion and aggregation in response to adrenaline, sodium arachidonate, U44069, PAF-acether, A23187 and low concentrations of collagen. Thrombin and higher concentrations of collagen produce a normal response. Secretion of β-thromboglobulin and platelet factor 4 parallels that of 5-HT. Thromboxane B₂ is produced normally in response to exogenous arachidonate and to stimulation by thrombin, collagen and A23187 in all concentrations tested. The patient's endoperoxides and thromboxane A₂ aggregate aspirin-treated platelets, though his platelets are themselves unresponsive. Cyclic AMP is present at normal concentration in the patient's unstimulated platelet-rich plasma, and PGI₂ inhibits platelet aggregation by ADP and thrombin in a normal dose-related manner. Platelet ultrastructure, 5-HT uptake and content of adenine nucleotides, platelet factor 4 and β-thromboglobulin are all within normal limits. When the patient's platelets were loaded with the fluorescent dye quin 2, which serves as an indicator of cytoplasmic free calcium ions, their responses to thrombin, whether in the presence or virtual absence of extracellular Ca2+, were entirely normal in respect of free calcium ions, secretion, shape-change and aggregation. In response to ionomycin, however, a normal increase in free calcium ions was accompanied by normal shape-change but virtually no aggregation or 5-HT secretion. The platelet calmodulin content was normal. These findings show that the defect in this patient's platelets is of utilization of cytoplasmic Caz+ for secretion and aggregation, rather than of Ca2+ uptake or mobilization of Ca2+ from intracellular storage sites. It is suggested that the most likely site of the defect is the phosphorylation of one of the proteins concerned in the secretory mechanism.     

Tec regulates platelet activation by GPVI in the absence of Btk

The Tec family kinase Btk plays an important role in the regulation of phospholipase C gamma 2 (PLC gamma 2) downstream of the collagen receptor glycoprotein VI (GPVI) in human platelets. Platelets also express a second member of this family, Tec; however, its function has not been analyzed. To address the role of Tec, we analyzed Btk-/-, Tec-/-, and Btk/Tec double-deficient (Btk-/-/Tec-/-) platelets. Tec-/- platelets exhibit a minor reduction in aggregation to threshold concentrations of collagen or the GPVI-specific agonist collagen-related peptide (CRP), whereas responses to higher concentrations are normal. Tyrosine phosphorylation of PLC gamma 2 by collagen and CRP is not altered in Tec-/- platelets. However, Btk-/-/Tec-/- platelets exhibit a greater reduction in PLC gamma 2 phosphorylation than is seen in the absence of Btk, thus revealing an important role for Tec in this situation. Furthermore, Btk-/-/Tec-/- platelets fail to undergo an increase in Ca2+, aggregation, secretion, and spreading in response to collagen or CRP, whereas they aggregate normally to adenosine diphosphate (ADP) and spread on fibrinogen. A residual GPVI signal exists in the Btk-/-/Tec-/- platelets as CRP synergizes with ADP to mediate aggregation. These results demonstrate an essential requirement for Tec and Btk in platelet activation by GPVI and reveal a functional role for Tec in the regulation of PLC gamma 2 in the absence of Btk.     

Inhibitory mechanism of human platelet aggregation by nafamostat mesilate

We found that nafamostat mesilate (NM) inhibits platelet aggregation induced by all agonists tested, including ADP, collagen, arachidonic acid, thromboxane A analog, A23187, phorbol 12-myristate 13-acetate (PMA), NaF and thrombin. The IC50 values were in the range of 9.3-17.8 microM. NM inhibited agonists-induced aspirin-treated platelet aggregation at >10 microM, suggesting that the action site lies beyond thromboxane (TXA)2 formation. However, NM inhibited thrombin (0.5 IU/ml)-induced TXB2 formation (IC50 = 1.9 +/- 0.6 microM, mean +/- SD). Intracellular Ca2+ mobilization was also inhibited only when platelets were challenged by thrombin, but the effect was found at NM concentrations >50 microM. This finding suggests that NM reduces the responses to thrombin by inhibiting its proteolytic activity on the platelet thrombin receptor (PAR1). NM did not affect the intracellular cAMP concentration or A-kinase activity. Agonists-induced surface expression of activated glycoprotein (GP)IIb-IIIa was inhibited by 10 microM NM and was completely inhibited by 50 microM NM. Since this inhibitory effect was parallel to the inhibition of platelet aggregation, the main inhibitory mechanism of NM against platelet aggregation seemed to be the suppression of activated GPIIb-IIIa expression, which makes it able to bind fibrinogen.     

Inhibitory effect of Ginkgo biloba extract on human platelet aggregation

The effect of pure flavonoids and Gingko biloba extract (GBE) on human platelet aggregation was investigated. Most of the flavonoids and vitamin E did not affect platelet aggregation in platelet-rich plasma (PRP); however some of these flavonoids inhibited platelet aggregation in gel-filtered platelets (GFP). GBE inhibited both ADP- and collagen-induced platelet aggregation in PRP, GFP and in whole blood in a dose-dependent manner. GBE at very low concentrations inhibited whole blood aggregation induced by ADP compared with those used for PRP or GFP. Flavonoids and GBE decreased the production of TxA(2) induced by collagen and ADP in PRP. However, no correlation was observed between the inhibition of platelet aggregation and the decrease of TxA(2) synthesis. GBE and flavonoids did not affect platelet membrane fluidity. However, the incubation of PRP with GBE increased cAMP levels in platelets, which is known to inhibit platelet activation by lowering intracellular Ca2+ levels. GBE is a mixture of many compounds, including flavonoids and gingkoglides, which affect metabolism of cAMP, TxA(2) and Ca2+ in platelets. It is effective in the inhibition of platelet aggregation, both in PRP and whole blood, and thus may be potentially used as an effective oral anti-platelet therapeutic agent.     

Characterization of platelet function in cyclic hematopoietic dogs

Platelet aggregation to incremental doses of eight different platelet agonists (collagen, thrombin, platelet-activating factor [PAF], arachidonic acid [AA] plus epinephrine, the calcium ionophore A23187, ADP, phospholipase C [PLC], and 12-O-tetradecanoyl phorbol-13-acetate [TPA]) was compared in normal (N) and cyclic hematopoietic (CH) dogs. Platelet aggregation was defective with collagen, PAF, TPA, and possibly thrombin as agonists but normal when ADP, PLC, arachidonic acid plus epinephrine, and A23187 were used as agonists with CH platelets. In heterozygous CH dogs, platelet aggregation was intermediately defective when tested with collagen and PAF as agonists. Thromboxane B2 (TXB2) concentrations (mean +/- SD; pg/10(6) platelets), as measured by RIA, were similar in CH and normal dogs both prior to (CH: 7.6 +/- 7.0; N: 5.5 +/- 3.9) and after collagen stimulation (collagen: 141.3 +/- 42.5; 123.1 +/- 38.4). Granule storage pools of serotonin and platelet adenine nucleotides were markedly decreased in homozygous CH but not heterozygous CH dogs. Thrombin stimulated phosphorylation of 40- and 20-kd proteins in platelets from CH and normal dogs to an equal extent. However, collagen-stimulated phosphorylation of the 40- but not the 20-kd protein was significantly decreased in platelets from CH dogs. These data suggest that there is a biochemical defect in platelets from CH dogs that results in storage pool disease and decreased phosphorylation of a 40-kd protein.     

Inhibitory mechanism of human platelet aggregation by nafamostat mesilate

We found that nafamostat mesilate (NM) inhibits platelet aggregation induced by all agonists tested, including ADP, collagen, arachidonic acid, thromboxane A analog, A23187, phorbol 12-myrisate 13-acetate (PMA), NaF and thrombin. The IC50 values were in the range of 9.3-17.8 mu M. NM inhibited agonists-induced aspirin-treated platelet aggregation at >10 mu M, suggesting that the action site lies beyond thromboxane (TXA)2 formation. However, NM inhibited thrombin (0.5 IU/ml)-induced TXB2 formation (IC50 = 1.9 +/- 0.6 mu M, mean +/- SD). Intracellular Ca2+ mobilization was also inhibited only when platelets were challenged by thrombin, but the effect was found at NM concentrations >50 mu M. This finding suggests that NM reduces the responses to thrombin by inhibiting its proteolytic activity on the platelet thrombin receptor (PAR1). NM did not affect the intracellular cAMP concentration or A-kinase activity. Agonists-induced surface expression of activated glycoprotein (GP)IIb-IIIa was inhibited by 10 mu M NM and was completely inhibited by 50 mu M NM. Since this inhibitory effect was parallel to the inhibition of platelet aggregation, the main inhibitory mechanism of NM against platelet aggregation seemed to be the suppression of activated GPIIb-IIIa expression, which makes it able to bind fibrinogen.     

Impaired cytoplasmic ionized calcium mobilization in inherited platelet secretion defects

Defects in platelet cytoplasmic Ca++ mobilization have been postulated but not well demonstrated in patients with inherited platelet secretion defects. We describe studies in a 42-year-old white woman, referred for evaluation of easy bruising, and her 23-year-old son. In both subjects, aggregation and 14C-serotonin secretion responses in platelet-rich plasma (PRP) to adenosine diphosphate (ADP), epinephrine, platelet activating factor (PAF), arachidonic acid (AA), U46619, and ionophore A23187 were markedly impaired. Platelet ADP and adenosine triphosphate (ATP), contents and thromboxane synthesis induced by thrombin and AA were normal. In quin2-loaded platelets, the basal intracellular Ca++ concentration, [Ca++]i, was normal; however, peak [Ca++]i measured in the presence of 1 mmol/L external Ca++ was consistently diminished following activation with ADP (25 mumol/L), PAF (20 mumol/L), collagen (5 micrograms/mL), U46619 (1 mumol/L), and thrombin (0.05 to 0.5 U/mL). In aequorin-loaded platelets, the peak [Ca++]i studied following thrombin (0.05 and 0.5 U/mL) stimulation was diminished. Myosin light chain phosphorylation following thrombin (0.05 to 0.5 U/mL) stimulation was comparable with that in the normal controls, while with ADP (25 mumol/L) it was more strikingly impaired in the propositus. We provide direct evidence that at least in some patients with inherited platelet secretion defects, agonist-induced Ca++ mobilization is impaired. This may be related to defects in phospholipase C activation. These patients provide a unique opportunity to obtain new insights into Ca++ mobilization in platelets.     

Characterization of platelets from normal mink and mink with the Chediak-Higashi syndrome

Bleeding times of mink with the Chediak-Higashi (CH) syndrome was markedly prolonged. Platelet counts were normal but there was an impaired platelet aggregation response to collagen. The metabolic adenine nucleotide pool of platelets from normal and CH mink was labeled with 14C-adenine and the platelets were gel-filtered. Gel-filtered platelets (GFP) from CH mink contained only 37.9% of the adenosine triphosphate (ATP) and 9.6% of the adenosine diphosphate (ADP) found in normal platelets and the ATP/ADP ratio was similar to the 14C-ATP/14C-ADP ratio. Platelet content of Ca2+, Mg2+, and in particular 5-hydroxytryptamine was decreased. When GFP were incubated with thrombin to induce maximal secretion, only negligible amounts of ATP and ADP were released. The specific activity of the extracellular nucleotides approximated that within the platelet. These findings suggest that the stored nucleotide pool in CH platelets is virtually absent and that the abnormalities in platelet function may be due, in part, to the essential absence of secretable ADP and serotonin. The release of Ca2+ and Mg2+ by CH platelets was 56% and 27.8% of normal, respectively.     

Influence of hydroxyl radical scavengers on platelet function

The influence of four hydroxil radical (OH.) scavengers on platelet function was investigated. OH. scavengers inhibited ADP, collagen, arachidonic acid, PAF-induced platelet aggregation, and platelet cyclooxygenase pathway activation, which was studied by evaluating platelet malondialdehyde and serum thromboxane A2 formation. The latter was not affected by superoxide dismutase, catalase, or metal ion chelants such as desferioxamine or DETAPAC. The detection of deoxyribose degradation by stimulated platelets suggested that platelets produce OH.. This study shows that activated platelets produce free radicals and that antioxidant agents such as OH. scavengers inhibit platelet function.     

Inhibitory mechanisms of kinetin,a plant growth-promoting hormone,in platelet aggregation

Kinetin has been shown to have anti-aging effects on several different systems including plants and human cells. The aim of this study was to examine the detailed inhibitory mechanisms of kinetin in platelet aggregation. In this study, kinetin concentration-dependently (50-150 μM) inhibited platelet aggregation in human platelets stimulated by agonists. Kinetin (70 and 150 μM) also concentration-dependently inhibited intracellular Ca²⁺ mobilization and phosphoinositide breakdown in platelets stimulated by collagen (1 μg/ml). Kinetin (70 and 150 μM) significantly inhibited thromboxane A₂ formation stimulated by collagen (1 μg/ml) and arachidonic acid (60 μM) in human platelets. In addition, kinetin (70 and 150 μM) significantly increased the formation of cyclic AMP. Intracellular pH values were measured spectrofluorometrically using the fluorescent probe BCECF-AM in platelets. The thrombin-evoked increase in pHi was markedly inhibited in the presence of kinetin (70 and 150 μM). Rapid phosphorylation of a platelet protein of molecular weight (M_r) 47 000 (P47), a marker of protein kinase C activation, was triggered by collagen (1 μg/ml). This phosphorylation was inhibited by kinetin (70 and 150 μM). In conclusion, these results indicate that the anti-platelet activity of kinetin may be involved in the following pathways: kinetin's effects may initially be due to inhibition of the activation of phospholipase C and the Na⁺/H⁺ exchanger. This leads to lower intracellular Ca²⁺ mobilization, followed by inhibition of TxA₂ formation and then increased cyclic AMP formation, followed by a further inhibition of the Na⁺/H⁺ exchanger, ultimately resulting in markedly decreased intracellular Ca²⁺ mobilization and phosphorylation of P47. These results suggest that kinetin has an effective anti-platelet effect and that it may be a potential therapeutic agent for arterial thrombosis.     

Impaired platelet response to thromboxane-A2 and defective calcium mobilization in a patient with a bleeding disorder

Platelet aggregation, secretion, and thromboxane formation induced by various agonists, including arachidonate, prostaglandin-G2 (PGG2), and thromboxane-A2 (TxA2), were examined in a patient with a bleeding disorder who was previously reported to have a TxA2-related defect. Aggregation and 14C-5HT secretion were decreased, and no TxB2 formation occurred in response to adenosine diphosphate (ADP), epinephrine, or collagen. Arachidonate-induced aggregation and TxB2 formation, and PGG2- induced aggregation (but not TxB2 formation) were impaired at low agonist concentrations. The patient's platelets did not aggregate in response to TxA2 generated from arachidonate in normal platelets, but were capable of synthesizing TxA2 from both arachidonate and PGG2. In addition, aggregation and secretion induced by low concentrations of the ionophore A23187 were impaired in platelet-rich plasma (PRP) and in gel-filtered platelets in the absence of extracellular calcium; these responses became normal at higher A23187 concentrations or, in GFP, at low A23187 concentrations in the presence of exogenous calcium. These findings indicate that the TxA2 defect in this patient does not result from a thromboxane synthetase deficiency, but may be due to impaired mobilization of platelet calcium, and thus are consistent with the possibility that TxA2 may act as a calcium ionophore.     

Platelet secretion defect associated with impaired liberation of arachidonic acid and normal myosin light chain phosphorylation

We describe four patients with impaired platelet aggregation and 14C- serotonin secretion during stimulation with adenosine diphosphate (ADP), epinephrine, collagen, and platelet-activating factor. The response to arachidonic acid was normal in all patients with regard to aggregation and in three of the four with regard to 14C-serotonin secretion. The total platelet adenosine triphosphate (ATP) and ADP content and the ATP to ADP ratio was normal in all patients, thereby excluding storage pool deficiency as the cause of the secretion defect. Studies with 3H-arachidonic acid-labeled platelets revealed that the thrombin-induced liberation of arachidonic acid from membrane-bound phospholipids was impaired in these patients. Further, platelet thromboxane B2 production, measured using a radioimmunoassay, was diminished during stimulation with ADP and thrombin, but was normal with arachidonic acid, indicating that the oxygenation of arachidonic acid was normal and that the diminished thromboxane production was due to a defect in the liberation of arachidonic acid. Release of arachidonic acid is mediated by phospholipases that are Ca++ dependent. To examine whether these patients may have a defect in making intracellular Ca++ available, another Ca++-dependent process, myosin light chain phosphorylation, was studied during thrombin stimulation. Platelets from three of the patients were found to behave the same as normal ones, suggesting that the deficiency in phospholipase activity may not be due to impaired Ca++ mobilization. Our studies demonstrate a novel group of patients with platelet secretion defects associated with impaired liberation of arachidonic acid from phospholipids. These patients exemplify a congenital defect, other than deficiencies of cyclooxygenase and thromboxane synthetase, by which thromboxane production may be impaired in platelets.     

The P2 receptors in platelet function

After vessel wall injury, platelets adhere to the exposed subendothelium, are activated, and release mediators such as thromboxane A (2) (TXA (2)) and nucleotides stored at very high concentration in the so-called dense granules. Among other soluble agents, released nucleotides act in a positive feedback mechanism to cause further platelet activation and amplify platelet responses induced by agents such as thrombin or collagen. Adenine nucleotides act on platelets through three distinct P2 receptors: two are G protein-coupled adenosine diphosphate (ADP) receptors, namely the P2Y (1) and P2Y (12) receptor subtypes; the P2X (1) receptor ligand-gated cation channel is activated by adenosine triphosphate (ATP). The P2Y (1) receptor initiates platelet aggregation but is not sufficient for a full platelet aggregation in response to ADP, whereas the P2Y (12) receptor is responsible for completion of the aggregation to ADP. This receptor, the molecular target of the antithrombotic drug clopidogrel, is responsible for most of the potentiating effects of ADP when platelets are stimulated by agents such as thrombin, collagen, or immune complexes. The P2X (1) receptor is involved in platelet shape change and in activation by collagen under shear conditions. Each of these receptors is coupled to specific signal transduction pathways in response to ADP or ATP and is differentially involved in all of the sequential events involved in platelet function and hemostasis. As such, they represent potential targets for antithrombotic drugs.     

Human platelet signaling defect characterized by impaired production of inositol-1,4,5-triphosphate and phosphatidic acid and diminished Pleckstrin phosphorylation: evidence for defective phospholipase C activation

Signal transduction on platelet activation involves phosphoinositide- specific phospholipase C (PLC)-mediated hydrolysis of phosphatidylinositides and formation of inositol-1,4,5-triphosphate [I(1,4,5)P3], which mediates Ca2+ mobilization, and diacylglycerol (DG), which activates protein kinase C (PKC) to phosphorylate a 47-kD protein (Pleckstrin). We studied these events in two related patients previously reported (Blood 74:664, 1989) to have abnormal aggregation and 14C-serotonin secretion, and impaired intracellular Ca2+ mobilization in response to several agonists. Thrombin-induced I(1,4,5)P3 and phosphatidic acid formation were diminished. Pleckstrin phosphorylation was impaired on activation with thrombin, platelet- activating factor, and ionophore A23187, but was normal with PKC activator 1,2-dioctonyl-sn-glycerol (DiC8). Ca2+ mobilization induced by guanosine triphosphate (GTP) analog guanosine 5'-0-(3 thiotriphosphate) (GTP gamma S) was diminished. Pretreatment with either A23187 or DiC8 did not correct the impaired adenine diphosphate- induced secretion; however, upon stimulation with A23187 plus DiC8, pleckstrin phosphorylation and secretion were normal, indicating that both PKC activation and Ca2+ mobilization are essential for normal secretion. We conclude that these patients have a unique inherited platelet defect in formation of two key intracellular mediators [I(1,4,5)P3 and DG] and in the responses mediated by them due to a defect in postreceptor mechanisms of PLC activation.