Hereditary defect of the platelet ADP receptor(s)

Four patients with a previously unrecognized congenital disorder of platelet function have recently been described. Their platelets aggregate very poorly to exogenous ADP. The abnormality is likely due to a severe defect of the platelet ADP receptor that is coupled to adenylate cyclase, as suggested by the following findings: 1) ADP does not normally lower cAMP levels of PGE1-treated platelets; 2) platelet shape change induced by ADP is normal; 3) the binding of \[radiolabelled]ADP to formalin-fixed platelets or of the ADP analogue \[radiolabelled]2-MeS-ADP to fresh platelets is severely defective. Since all patients that have been described were born from consanguineous parents, the condition seems to be inherited as an autosomal recessive trait. Platelets of an obligate heterozygote have intermediate binding sites for 2-M eS-ADP, undergo a normal primary wave of aggregation induced by exogenous ADP, but do not normally secrete the content of their granules when stimulated by release-inducing agonists. Studies of normal platelets treated with acetylsalycilic acid revealed that ADP potentiates platelet secretion directly, and that the full complement of its platelet receptors appears to be necessary for this function.     

Hereditary defect of the platelet ADP receptor(s)

Four patients with a previously unrecognized congenital disorder of platelet function have recently been described. Their platelets aggregate very poorly to exogenous ADP. The abnormality is likely due to a severe defect of the platelet ADP receptor that is coupled to adenylate cyclase, as suggested by the following findings: 1) ADP does not normally lower cAMP levels of PGE1-treated platelets; 2) platelet shape change induced by ADP is normal; 3) the binding of [radiolabelled]ADP to formalin-fixed platelets or of the ADP analogue [radiolabelled]2-MeS-ADP to fresh platelets is severely defective. Since all patients that have been described were born from consanguineous parents, the condition seems to be inherited as an autosomal recessive trait. Platelets of an obligate heterozygote have intermediate binding sites for 2-M eS-ADP, undergo a normal primary wave of aggregation induced by exogenous ADP, but do not normally secrete the content of their granules when stimulated by release-inducing agonists. Studies of normal platelets treated with acetylsalycilic acid revealed that ADP potentiates platelet secretion directly, and that the full complement of its platelet receptors appears to be necessary for this function.     

Differential requirements for platelet aggregation and inhibition of adenylate cyclase by epinephrine. Studies of a familial platelet alpha 2-adrenergic receptor defect

We describe a family whose members have impaired platelet aggregation and secretion responses to epinephrine with normal responses to adenosine diphosphate and collagen. Platelet alpha 2-adrenergic receptors (measured using 3H methyl-yohimbine) were diminished in the propositus (78 sites per platelet), his two sisters (70 and 27 sites per platelet), and parents (37 and 63 sites per platelet), but not in two maternal aunts (12 normal subjects, 214 +/- 18 sites per platelet; mean +/- SE). However, the inhibition of cyclic adenosine monophosphate (cAMP) levels by epinephrine in platelets exposed to 400 nmol/L PGI2 was similar in the patients and five normal subjects (epinephrine concentration for 50% inhibition, 0.04 +/- 0.01 mumol/L v 0.03 +/- 0.01 mumol/L; P greater than .05). In normal platelets, the concentration of yohimbine (0.18 mumol/L) required for half maximal inhibition of aggregation induced by 2 mumol/L epinephrine was lower than that for inhibition of its effect on adenylate cyclase (1.6 mumol/L). In quin2 loaded platelets, thrombin (0.1 U/mL) stimulated rise in cytoplasmic Ca2+ concentration, [Ca2+]i, was normal in the two patients studied. The PGI2 analog ZK 36,374 completely inhibited thrombin-induced rise in [Ca2+]i; the reversal of this inhibition by epinephrine was normal in the two patients. Thus, despite the impaired aggregation response to epinephrine, platelets from these patients have normal ability to inhibit PGI2-stimulated cAMP levels. These patients with an inherited receptor defect provide evidence that fewer platelet alpha 2-adrenergic receptors are required for epinephrine-induced inhibition of adenylate cyclase than for aggregation.     

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.     

P2Y12, a new platelet ADP receptor, target of clopidogrel

Clopidogrel is a potent antithrombotic drug that inhibits ADP-induced platelet aggregation. The results of large clinical trials have demonstrated an overall benefit of clopidogrel over aspirin in the prevention of vascular ischemic events (myocardial infarction, stroke, vascular death) in patients with a history of symptomatic atherosclerotic disease. The antiaggregating effect of clopidogrel is attributed to an irreversible inhibition of ADP binding to a purinergic receptor present at the platelet surface. Clopidogrel is not active in vitro and can be considered a precursor of an active metabolite formed in the liver. The chemical structure of this active metabolite and its biological activity have been described recently. Several purinergic receptors have been described on platelets; P2X (1), a calcium channel, and P2Y1 a Gq-coupled seven-transmembrane domain receptor, have been found not to be antagonized by clopidogrel. Another Gi (2)-coupled receptor (named P2Y12) has been recently cloned and stably expressed in CHO cells. These cells displayed a strong affinity for (33)P-2MeS-ADP, a stable analogue of ADP, the binding characteristics of which corresponded in all points to those observed on platelets. The binding of (33)P-2MeS-ADP to these cells was strongly inhibited by the active metabolite of clopidogrel with a potency that was consistent with that observed for this compound on platelets. In these transfected CHO cells, as in platelets, ADP and 2MeS-ADP induced adenylyl cyclase downregulation, an effect that was inhibited by the active metabolite of clopidogrel. These results demonstrate that this receptor corresponds to the previously called "P2t" platelet receptor and show that the active metabolite of clopidogrel binds in a covalent manner to this receptor, thus explaining how it blocks the aggregating effect of ADP on platelets.     

Platelet aggregation and adenosine diphosphate/adenosine triphosphate receptors: a historical perspective

The work of many investigators since adenosine diphosphate (ADP) was recognized as a platelet aggregating agent in 1961 has led to an appreciation of the important part that ADP plays in hemostasis and thrombosis. Recently, interest has focused on the platelet receptors for ADP and adenosine triphosphate (ATP). Platelets are unique because they have two P2Y receptors that must act in concert to achieve a normal aggregation response. The P2Y (1) receptor is responsible for mobilizing internal calcium, platelet shape change, and weak aggregation. The P2Y (12) receptor inhibits adenylyl cyclase, but the concentration of cyclic AMP is reduced only if it has been raised from its low basal levels by stimulation of adenylyl cyclase by an aggregation inhibitor such as adenosine or prostaglandin I (2). The abnormal bleeding of the rare patients whose platelets lack P2Y (12) and the beneficial clinical effects of ticlopidine and clopidogrel that block this receptor indicate that P2Y (12), in addition to inhibiting adenylyl cyclase, may have an as yet unidentified role that is needed for its cooperative aggregation effect with P2Y (1). ATP stimulates a rapid influx of calcium into platelets through the P2X (1) receptor, and it may synergize with ADP when these two nucleotides are released from platelets at a site of vessel injury.     

The P2 receptors and congenital platelet function defects

Platelets possess three P2 receptors: two (P2Y (1) and P2Y (12)) are receptors for adenosine diphosphate (ADP), and one (P2X1) is a receptor for adenosine triphosphate (ATP). The P2Y (1) receptor, which is coupled to Gq and phospholipase C-beta, is responsible for mobilization of ionized calcium from internal stores and mediates the ADP-induced platelet shape change and initial wave of rapidly reversible aggregation. The other ADP receptor, P2Y (12), is negatively coupled to adenylyl cyclase through Gi and mediates a progressive and sustained ADP-induced aggregation not preceded by shape change. In addition, this receptor plays an important role in the potentiation of platelet secretion induced by several platelet agonists. The combined action of P2Y (1) and P2Y (12) is necessary for the full platelet aggregation response to ADP. Four patients with severe deficiency of P2Y (12) have been described so far. Sequence analysis of the P2Y (12) locus of three of these patients revealed homozygous mutations that produced a frame shift mutation and premature truncation of the protein. The fourth patient had an allele with a frame shift mutation and a normal allele, which could be silenced by an additional, as yet unknown, mutation. More recently, we described a patient with a congenital bleeding disorder and a dysfunctional P2Y (12). The patient is a compound heterozygote, in whom one allele contained a G to A transition resulting in an Arg (256) to Gln codon substitution (R256Q) and the other allele contained a C to T transition resulting in an Arg (265) to Trp codon substitution (R265W). The two substitutions are located in TM6 and EL3 of the receptor. Stable Chinese hamster ovaries (CHO) cell lines were established expressing either wild-type P2Y (12) and P2Y (12)(R256Q) or P2Y (12)(R265W). Neither mutation blocked the ability of the P2Y (12) receptor to translocate to the CHO cell surface. ADP at all tested concentrations (0.1 to 10 muM) greatly inhibited the forskolin-induced increase of cyclic adenosine monophosphate (cAMP) in CHO cells transfected with wild-type P2Y (12), whereas CHO cells transfected with either mutant protein were only partially inhibited by ADP. Thus, the molecular basis for the patient's dysfunctional platelet phenotype is explained by missense mutations and the expression of a dysfunctional P2Y (12) receptor. The localization of both mutations in TM6 and EL3 identifies this region of P2Y (12) as a structurally and functionally critical region of the receptor.     

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.     

Patients with a prolonged bleeding time and normal aggregation tests may have storage pool deficiency: studies on one hundred six patients

One hundred six patients with storage pool deficiency (SPD) were studied with respect to platelet count, bleeding time, total platelet ATP and ADP, platelet serotonin, and in vitro aggregation. The diagnosis of SPD was made on basis of a prolonged bleeding time, a decreased total platelet ADP, and a diminished level of serotonin. Fifty-one patients from 34 unrelated families had congenital SPD, and 55 patients had acquired SPD. Congenital SPD was a common disorder in patients with a lifelong bleeding tendency and a prolonged bleeding time. The frequency in this group of patients was 18%, about one-half the frequency of von Willebrand's disease (vWd). Twenty-three percent of all patients had normal aggregation responses to ADP, epinephrine, and collagen; 33% had aggregation tracings typical for a secretion defect; and 44% had miscellaneous aggregation abnormalities. These findings indicate that SPD is common, heterogeneous, and not necessarily associated with in vitro aggregation abnormalities.     

Reciprocal cross-talk between P2Y1 and P2Y12 receptors at the level of calcium signaling in human platelets

Adenosine diphosphate (ADP), an important platelet agonist, acts through 2 G-protein-coupled receptors (GPCRs), P2Y(1) and P2Y(12), which signal through Gq and Gi, respectively. There is increasing evidence for cross-talk between signaling pathways downstream of GPCRs and here we demonstrate cross-talk between these 2 ADP receptors in human platelets. We show that P2Y(12) contributes to platelet signaling by potentiating the P2Y(1)-induced calcium response. This potentiation is mediated by 2 mechanisms: inhibition of adenylate cyclase and activation of phosphatidylinositol 3 (PI 3)-kinase. Furthermore, the Src family kinase inhibitor PP1 selectively potentiates the contribution to the calcium response by P2Y(12), although inhibition of adenylate cyclase by P2Y(12) is unaffected. Using PP1 in combination with the inhibitor of PI 3-kinase LY294002, we show that Src negatively regulates the PI 3-kinase-mediated component of the P2Y(12) calcium response. Finally, we were able to show that Src kinase is activated through P2Y(1) but not P2Y(12). Taken together, we present evidence for a complex signaling interplay between P2Y(1) and P2Y(12), where P2Y(12) is able to positively regulate P2Y(1) action and P2Y(1) negatively regulates this action of P2Y(12). It is likely that this interplay between receptors plays an important role in maintaining the delicate balance between platelet activation and inhibition during normal hemostasis.     

Reference curves for aggregation and ATP secretion to aid diagnose of platelet-based bleeding disorders: effect of inhibition of ADP and thromboxane A(2) pathways

Platelet aggregation is widely used in clinical laboratories to evaluate patients with bleeding disorders of suspected platelet aetiology. Simultaneous monitoring of ATP release as a measure of dense granule secretion provides additional information to aid diagnosis. There is, however, no standard way of performing or interpreting these tests. The present study has evaluated aggregation and ATP secretion to eight platelet agonists in healthy donors and has evaluated the reproducibility of response for a number of variables, including platelet number and time after donation. The effect of inhibition of the two major platelet feedback mediators, ADP and thromboxane A(2) (TxA(2)), was investigated using the P2Y(1) and P2Y(12) receptor antagonists, MRS2179 and AR-C67085, and the cyclooxygenase inhibitor, indomethacin. The results demonstrate that, if used within certain boundaries, the investigation of platelet aggregation and secretion is a powerful way to discriminate between differing pathways of platelet activation. The present data-set are an invaluable resource to the clinical laboratory to aid evaluation of patients with suspected platelet-based bleeding disorders.     

Qualitative platelet defects in chronic myeloproliferative disorders: evidence for reduced ATP secretion

19 consecutive untreated patients with chronic myeloproliferative disorders and thrombocytosis were subjected to comprehensive platelet function tests including platelet aggregometry. 12 patients had essential thrombocythaemia (ET) and 7 patients had polycythaemia vera (PV). Bleeding time was normal. Arachidonic acid, collagen and ristocetin aggregation were abnormal only in a minority of patients, whereas ADP aggregation was impaired in 16 out of 19 patients. The most conspicuous findings were abolished second-wave adrenalin aggregation, increased ADP aggregation threshold, and markedly reduced ATP secretion during collagen-induced aggregation. This triad of qualitative platelet defects seems to be a good diagnostic marker of chronic myeloproliferative disease with thrombocytosis.     

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.     

Combined defect in membrane expression and activation of platelet GPIIb--IIIa complex without primary sequence abnormalities in myeloproliferative disease

Defects in glycoprotein (GP)IIb-IIIa or in its activation may cause abnormal platelet aggregation and a bleeding diathesis. We report studies in a 67-year-old man with a myeloproliferative disease and markedly abnormal platelet responses. By flow cytometry, platelet binding of two complex-specific anti-GPIIb-IIIa monoclonal antibodies (mAbs), A2A9 and 10E5, was approximately 50% of normal. An enzyme-linked immunosorbent assay (ELISA) using immobilized kistrin showed 18% of normal membrane GPIIb-IIIa complex. By immunoblot analysis, GPIIb and GPIIIa levels in platelet lysates and membranes were near normal. Activation of GPIIb-IIIa, monitored with mAb PAC-1, was markedly decreased (< 20% of normal) in response to ADP, thrombin and platelet-activating factor (PAF); expression of ligand-induced binding sites (LIBS) was < or = 30% of normal. Signal transduction-independent LIBS expression, induced by echistatin, was approximately 60% of normal, suggesting that the integrin present had intact ligand-binding capability. Sequence analysis of GPIIb and GPIIIa cDNA, and platelet mRNA levels for both subunits, were normal. These findings document an acquired combined defect in membrane expression (secondary to a defect in post-translational processing of the complex) and inside-out signalling-dependent activation of the GPIIb-IIIa complex.     

Absence of compensatory platelet activation in patients with severe haemophilia,but evidence for a platelet collagen-activation defect

Severe haemophilia is a serious, haemorrhagic disorder of the plasmatic coagulation system. In this study we investigated, whether 'compensatory' activation of the platelet coagulation system occurs in this situation. Platelet function was investigated with aggregation, adhesion and flow cytometric assays. In addition, we performed clot and platelet plug formation tests and determined endogenous thrombin potentials in patients with severe haemophilia A or B; results were compared to those of healthy controls. Platelet aggregation in response to stimulation with ADP, ristocetin and epinephrine was similar in patients and controls; aggregation in response to collagen was reduced significantly in haemophiliacs. Flow cytometric analysis of P-selectin (CD 62P) and CD 63, of the conformationally changed GP IIb/IIIa with PAC 1 and of thrombospondin bound to CD 36 (GP IV) was performed at baseline and post stimulation. Baseline expression of all markers was similar in haemophiliacs and controls. After stimulation of the platelet thrombin receptors with the thrombin receptor activating peptide (TRAP) 6, the surface expression of all markers increased significantly; again, the expression was similar in haemophiliacs and controls. With thrombelastography and PFA 100 analysis, clot formation under low shear and platelet plug formation under high shear is measured. Both test results revealed a significantly reduced clot and platelet plug formation capacity in severe haemophiliacs. Our results did not reveal signs of enhanced platelet preactivation in haemophiliacs, indicating that baseline platelet reactivity in severe haemophilia remains in a neutral state, despite the severely haemorrhagic condition. As expected, both thrombin and clot formation capacities were impaired significantly in severe haemophilia. The reduced response to collagen-based platelet stimulation tests is indicative of a concomitant platelet function defect. This defect probably contributes to the intensity of bleeding events in patients with severe haemophilia.     

Absence of compensatory platelet activation in patients with severe haemophilia,but evidence for a platelet collagen-activation defect

Severe haemophilia is a serious, haemorrhagic disorder of the plasmatic coagulation system. In this study we investigated, whether 'compensatory' activation of the platelet coagulation system occurs in this situation. Platelet function was investigated with aggregation, adhesion and flow cytometric assays. In addition, we performed clot and platelet plug formation tests and determined endogenous thrombin potentials in patients with severe haemophilia A or B; results were compared to those of healthy controls. Platelet aggregation in response to stimulation with ADP, ristocetin and epinephrine was similar in patients and controls; aggregation in response to collagen was reduced significantly in haemophiliacs. Flow cytometric analysis of P-selectin (CD 62P) and CD 63, of the conformationally changed GP IIb/IIIa with PAC 1 and of thrombospondin bound to CD 36 (GP IV) was performed at baseline and post stimulation. Baseline expression of all markers was similar in haemophiliacs and controls. After stimulation of the platelet thrombin receptors with the thrombin receptor activating peptide (TRAP) 6, the surface expression of all markers increased significantly; again, the expression was similar in haemophiliacs and controls. With thrombelastography and PFA 100® analysis, clot formation under low shear and platelet plug formation under high shear is measured. Both test results revealed a significantly reduced clot and platelet plug formation capacity in severe haemophiliacs. Our results did not reveal signs of enhanced platelet preactivation in haemophiliacs, indicating that baseline platelet reactivity in severe haemophilia remains in a neutral state, despite the severely haemorrhagic condition. As expected, both thrombin and clot formation capacities were impaired significantly in severe haemophilia. The reduced response to collagen-based platelet stimulation tests is indicative of a concomitant platelet function defect. This defect probably contributes to the intensity of bleeding events in patients with severe haemophilia.     

Platelet adhesion defect in type I Gaucher Disease is associated with a risk of mucosal bleeding

Patients with type I Gaucher Disease (GD) may have a clinically significant bleeding tendency that is disproportionate to their platelet count. We hypothesized that impaired platelet adhesion might contribute to bleeding tendency. Adult patients with type I GD with platelet counts ≥130×10(9) /l and haematocrit ≥30% (n=48), obligatory carriers (n=52), and healthy controls (n=19) were studied. Platelet adhesion, using the IMPACT-R (Cone and Plate(let) Analyser), and platelet aggregation were determined. Type I GD patients had significantly lower platelet adhesion [surface coverage %, median (interquartile range)] 4·6 (3·2-7·5), compared to controls, 8·7 (7·6-10·3), or carriers, 8·1 (6·5-9·4; P=0·001). Platelet adhesion was not affected by the use of disease-specific enzyme replacement therapy but was improved in patients after splenectomy, 7·2 (5·8-9·3). Mixing tests showed that the reduced adhesion was an intrinsic platelet defect. Mucosal bleeding was reported in 17 (35·4%) patients and was associated with abnormal adhesion [P=0·037, with an Odds Ratio (95% confidence interval) of 5·73 (1·1-29·6)]. Five patients (22%) had reduced platelet aggregation, all of whom had reduced platelet adhesion. Platelet aggregation defect was not associated with mucosal bleeding. In conclusion, platelet adhesion defect is a major thrombocytopathy in type I GD patients and can explain part of the increased tendency to bleeding.     

Diminished platelet adenylate cyclase activation by prostaglandin D2 in acute thrombosis.

Prostaglandin D2 (PGD2) produced by platelets can inhibit aggregation via activation of platelet adenylate cyclase. PGD2 activation of platelet cyclase in platelet membrane fractions was studied in 20 consecutive patients hospitalized with acute deep-vein thrombosis and/or pulmonary embolism. In nine patients, PGD2-stimulated enzyme activity was decreased at all concentrations of PGD2 studied. This altered enzyme sensitivity was specific for PGD2 as basal enzyme activity, and prostaglandin E1, prostaglandin I2, and sodium fluoride stimulated adenylate cyclase was normal. The effect of PGD2 on platelet aggregation and 14C-serotonin release was also studied in one patient where a four-fold higher concentration of PGD2 was required to inhibit collagen-induced 14C-serotonin release. Binding studies using [3H]PGD2 as a radioligand indicated that this patient's platelets bound 10 fmole PGD2/10(8) platelets compared to 30 fmole/10(8) platelets in a normal control. Five patients had follow-up studies between 2 and 7 mo after their acute thrombotic event, and PGD2-stimulated adenylate cyclase activity returned towards normal in four. Since PGD2 is synthesized in platelets at concentrations sufficient to inhibit aggregation and activate adenylate cyclase, diminished platelet sensitivity to this prostaglandin could result in "hyperactivity" and contribute to the thrombosis observed in these patients.     

Heterogeneous defects of platelet secretion and responses to weak agonists in patients with bleeding disorders

Eleven patients with mild bleeding disorders had as a common abnormality, impaired platelet aggregation and secretion with low concentrations (0.5-1.0 micrograms/ml) of collagen and, in most cases, an absence of second phase aggregation with epinephrine. Platelet granule contents were normal, ruling out storage pool deficiency. To characterize further the platelet abnormalities, we measured aggregation, 14C-5HT secretion, and TxB2 formation induced by a variety of platelet agonists. In eight of the 11 patients we observed decreased initial rates as well as extents of aggregation with one or more weak agonists (ADP, epinephrine, thromboxane A2 and the endoperoxide analogue U44069), i.e. agonists which induced secretion only as a result of aggregation, but normal responses to strong agonists such as arachidonate and high (10 micrograms/ml) concentrations of collagen, which can induce secretion in the presence or absence of aggregation. In all of these patients, TxB2 formation with arachidonate and all concentrations of collagen was normal. The platelet defects in these eight patients have been designated as weak agonist response defects (WARDs). In contrast, the initial aggregation responses to all weak agonists were normal in the three other patients, while secretion and TxB2 formation induced by strong agonists were impaired. Thus, in contrast to the eight patients above, the platelet defects in these three patients were characteristic of defects in the secretion response per se. The results obtained in the 11 patients studied indicate that these types of platelet disorders, previously referred to as primary secretion defects, include defects in the initial platelet responses which precede secretion (WARD) as well as defects in the secretory mechanism per se. Both groups of defects appear to be heterogeneous in nature.     

Abnormal inside-out signal transduction-dependent activation of glycoprotein IIb-IIIa in a patient with impaired pleckstrin phosphorylation

Platelet-agonist interaction results in activation of glycoprotein (GP) IIb-IIIa complex and fibrinogen binding, a prerequisite for platelet aggregation. Fibrinogen binding exposes new antibody binding sites on GPIIb-IIIa (ligand-induced binding sites: LIBS). Signal transduction events, including pleckstrin phosphorylation by protein kinase C (PKC), are considered to regulate GPIIb-IIIa activation. We studied a 16-year- old white male with lifelong mucocutaneous bleeding manifestations and abnormal platelet aggregation and secretion in response to multiple agonists. Pleckstrin phosphorylation was diminished in response to platelet-activating factor (PAF; 4 and 400 nmol/L) and thrombin (0.05 U/mL). Binding of monoclonal antibodies (MoAbs) 10E5 and A2A9, which bind to both resting and activated GPIIb-IIIa, was normal. Binding of MoAb PAC1, which binds to only activated GPIIb-IIIa, was diminished upon activation with PAF, adenosine diphosphate (ADP), thrombin receptor agonist peptide (SFLLRN), A23187, and 1,2-dioctonylglycerol (DiC8). Signal transduction-dependent LIBS expression (studied using MoAb 62) induced by ADP, SFLLRN, and DiC8 and signal transduction- independent LIBS expression induced by RGDS peptide or disintegrin albolabrin were normal or minimally decreased, indicating the presence of intact ligand binding sites. We conclude that the patient's platelets have a defect in inside-out signal transduction-dependent GPIIb-IIIa activation due to an upstream defect in the signal transduction mechanisms rather than in the GPIIb-IIIa complex itself. Our findings extend the spectrum of congenital mechanisms leading to impaired aggregation from defects in GPIIb-IIIa per se to aberrations in signaling mechanisms.