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Trastornos plaquetarios primarios en la especie humana: Aspectos biomédicos: biología, patobiología y bioclínica
Abstract
Las enfermedades genéticas plaquetarias son desórdenes heterogéneos, algunos de ellos muy raros, que se presentan en la medicina clínica, caracterizados por trobocitopenia, plaquetas grandes (macrotrombocitopenias) y signos variables de hemorragia, así como trombosis en otros casos. La patogénesis y patofisiología es bastante desconocida y el propósito de este artículo es proveer una estructura lógica que resuma el conocimiento actual. Abreviaturas: PQ, plaquetas.
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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.
Platelets are specialized blood cells that play central roles in physiologic and pathologic processes of hemostasis, inflammation, tumor metastasis, wound healing, and host defense. Activation of platelets is crucial for platelet function that includes a complex interplay of adhesion and signaling molecules. This article gives an overview of the activation processes involved in primary and secondary hemostasis, for example, platelet adhesion, platelet secretion, platelet aggregation, microvesicle formation, and clot retraction/stabilization. In addition, activated platelets are predominantly involved in cross talk to other blood and vascular cells. Stimulated “sticky” platelets enable recruitment of leukocytes at sites of vascular injury under high shear conditions. Platelet-derived microparticles as well as soluble adhesion molecules, sP-selectin and sCD40L, shed from the surface of activated platelets, are capable of activating, in turn, leukocytes and endothelial cells. This article focuses further on the new view of receptor-mediated thrombin generation of human platelets, necessary for the formation of a stable platelet-fibrin clot during secondary hemostasis. Finally, special emphasis is placed on important stimulatory and inhibitory signaling pathways that modulate platelet function.
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.
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.
Recent advances in molecular genetics have revealed the mechanisms underlying a variety of inherited human disorders. Among them, mutations in G protein-coupled receptors have clearly demonstrated two types of abnormalities, namely loss of function and constitutive activation of the receptors. Thromboxane A2 (TXA2) receptor is a member of the family of G protein-coupled receptors and performs an essential role in hemostasis by interacting with TXA2 to induce platelet aggregation. Here we identify a single amino acid substitution (Arg60-->Leu) in the first cytoplasmic loop of the TXA2 receptor in a dominantly inherited bleeding disorder characterized by defective platelet response to TXA2. This mutation was found exclusively in affected members of two unrelated families with the disorder. The mutant receptor expressed in Chinese hamster ovary cells showed decreased agonist-induced second messenger formation despite its normal ligand binding affinities. These results suggest that the Arg60 to Leu mutation is responsible for the disorder. Moreover, dominant inheritance of the disorder suggests the possibility that the mutation produces a dominant negative TXA2 receptor.
G proteins play a major role in signal transduction upon platelet activation. We have previously reported a patient with impaired agonist-induced aggregation, secretion, arachidonate release, and Ca2+ mobilization. Present studies demonstrated that platelet phospholipase A2 (cytosolic and membrane) activity in the patient was normal. Receptor-mediated activation of glycoprotein (GP) IIb-IIIa complex measured by flow cytometry using antibody PAC-1 was diminished despite normal amounts of GPIIb-IIIa on platelets. Ca2+ release induced by guanosine 5'-[gamma-thio]triphosphate (GTP[gammaS]) was diminished in the patient's platelets, suggesting a defect distal to agonist receptors. GTPase activity (a function of alpha-subunit) in platelet membranes was normal in resting state but was diminished compared with normal subjects on stimulation with thrombin, platelet-activating factor, or the thromboxane A2 analog U46619. Binding of 35S-labeled GTP[gammaS] to platelet membranes was decreased under both basal and thrombin-stimulated states. Iloprost (a stable prostaglandin I2 analog) -induced rise in cAMP (mediated by Galphas) and its inhibition (mediated by Galphai) by thrombin in the patient's platelet membranes were normal. Immunoblot analysis of Galpha subunits in the patient's platelet membranes showed a decrease in Galphaq (<50%) but not Galphai, Galphaz, Galpha12, and Galpha13. These studies provide evidence for a hitherto undescribed defect in human platelet G-protein alpha-subunit function leading to impaired platelet responses, and they provide further evidence for a major role of Galphaq in thrombin-induced responses.
A form of autosomal dominant macrothrombocytopenia is characterized by mild or no clinical symptoms, normal platelet function, and normal megakaryocyte count. Because this condition has so far received little attention, patients are subject to misdiagnosis and inappropriate therapy. To identify the molecular basis of this disease, 12 Italian families were studied by linkage analysis and mutation screening. Flow cytometry evaluations of platelet membrane glycoproteins (GPs) were also performed. Linkage analysis in 2 large families localized the gene to chromosome 17p, in an interval containing an excellent candidate, the GPIbalpha gene. GPIbalpha, together with other proteins, constitutes the plasma von Willebrand factor (vWF) receptor, which is altered in Bernard-Soulier syndrome (BSS). In 6 of 12 families, a heterozygous Ala156Val missense substitution was identified. Platelet membrane GP studies were performed in 10 patients. Eight were distinguished by a reduction of GPs comparable to that found in a BSS heterozygous condition, whereas the other 2, without the Ala156Val mutation, had a normal content of platelet GPs. In conclusion, the current study provides evidence that most (10 of 12) patients with an original diagnosis of autosomal dominant macrothrombocytopenia shared clinical and molecular features with the heterozygous BSS phenotype. The remaining 2 affected subjects represented patients with "true" autosomal dominant macrothrombocytopenia; the GPIb/IX/V complex was normally distributed on the surface of their platelets. Thus, the diagnosis of heterozygous BSS must always be suspected in patients with inherited thrombocytopenia and platelet macrocytosis.
Platelet aggregation at sites of vascular injury is essential for the formation of the primary haemostatic plug. The mechanism of platelet aggregation under conditions of physiological flow is a complex multistep process, which requires the synergistic action of several different platelet receptors. Platelet interaction with collagen at sites of damage to the vascular endothelium involves adhesion, activation, secretion of platelet granular contents and finally aggregation. Other agonists other than collagen, such as fibrinogen, vWF and soluble agonists released from activated platelets (thromboxane A2 (TXA2) and ADP) are involved in platelet aggregation. Platelets express a variety of receptors including GP Ib-IX-V, GP VI, GP Ia-IIa and GP IIb-IIIa. One aspect of this complexity of function is the variety of inherited defects of platelet function. Hereditary disorders of platelet adhesion are Bernard-Soulier syndrome and von Willebrand disease. Glanzmann thrombasthenia is an inherited disorder of platelet aggregation. The application of molecular biology to the study of platelet disorders has identified defects in other collagen receptors, ADP receptors and TXA2 receptors. Defects affecting TXA2 production, the generation of procoagulant activity and secretion from dense bodies and alpha-granules are also encountered. Other rare diseases, Chediak-Higashi, Hermansky-Pudlak and Wiskott-Aldrich syndrome also affect platelet storage granules. In this article, recent advances in the understanding of platelet function and knowledge of inherited disorders that affect platelet adhesion and aggregation is reviewed. As progress advances towards individualisation of therapy the phenotypic bleeding tendency of each patient becomes relevant.
The Wiskott-Aldrich Syndrome (WAS) is an inherited immunodeficiency caused by a variety of mutations in the gene encoding the WAS protein (WASp). WASp is expressed in hematopoetic cells and facilitates the reorganization of the actin cytoskeleton in response to many important cell stimuli. Extensive study of WAS and more recently WASp has given great insight into the relevance of this molecule and related molecules to both basic cell biology and human immune defenses.
In the majority of patients with an inherited abnormality in platelet function and a bleeding diathesis, the underlying platelet molecular mechanisms are unknown. The usually considered entities, such as thrombasthenia, the Bernard-Soulier syndrome, and storage pool deficiency, occur in a small proportion of patients. A substantial number of patients present with decreased aggregation and secretion of dense granule contents upon activation, and are lumped in the category of primary secretion defects or platelet activation defects. Evidence is now available that defects in platelet signaling mechanisms may be the basis for the platelet dysfunction in some of these patients. This evidence is presented here. If the key components in signal transduction are the surface receptors, the G-proteins, and the effectors, evidence now exists for specific human platelet abnormalities at each of these levels. There is a pressing need for a concerted effort to delineate the molecular mechanisms in the large group of patients with impaired platelet function who represent an untapped reservoir of new information into normal platelet function.
Scott syndrome (SS) is a bleeding disorder characterized by a failure to expose phosphatidylserine (PS) to the outer leaflet of the platelet plasma membrane. Because the adenosine triphosphate (ATP)-binding cassette transporter A1 (ABCA1) is implicated in the exofacial translocation of PS, we assessed its role in the pathophysiology of a patient with SS. Substantially reduced levels of ABCA1 mRNA were found in the patient's leukocytes, compared with controls. The SS patient was heterozygous for a novel missense mutation c.6064G>A (ABCA1 R1925Q), absent from unaffected family members and controls. Both mutant and wild-type alleles were reduced in mRNA expression, and no causative mutation for this phenomenon was identified in the ABCA1 gene or its proximal promoter, suggesting a putative second mutation in a trans-acting regulatory gene may also be involved in the disorder in this patient. In vitro expression studies showed impaired trafficking of ABCA1 R1925Q to the plasma membrane. Overexpression of wild-type ABCA1 in SS lymphocytes complemented the Ca2+-dependent PS exposure at the cell surface. These data identify a mutation in ABCA1 that contributes to the defective PS translocation phenotype in our patient with SS.
Platelets from a patient with a mild inherited bleeding disorder and abnormal platelet aggregation and secretion show reduced generation of inositol 1,4,5-trisphosphate, mobilization of intracellular Ca2+, and phosphorylation of pleckstrin in response to several G protein mediated agonists, suggesting a possible defect at the level of phospholipase C (PLC) activation (see accompanying report). A procedure was developed that allows quantitation of platelet PLC isozymes. After fractionation of platelet extracts by high-performance liquid chromatography, 7 out of 10 known PLC isoforms were detected by immunoblot analysis. The amount of these isoforms in normal platelets decreased in the order PLC- gamma 2 > PLC-beta 2 > PLC-beta 3 > PLC-beta 1 > PLC-gamma 1 > PLC- delta 1 > PLC-beta 4. Compared with normal platelets, platelets from the patient contained approximately one-third the amount of PLC-beta 2, whereas PLC-beta 4 was increased threefold. These results suggest that the impaired platelet function in the patient in response to multiple G protein mediated agonists is attributable to a deficiency of PLC-beta 2. They document for the first time a specific PLC isozyme deficiency in human platelets and provide an unique opportunity to understand the role of different PLC isozymes in normal platelet function.
A patient with a mild bleeding disorder whose platelets responded defectively to thromboxane A2 (TXA2) was identified, and the mechanism of this dysfunction was analyzed. The platelets were defective in shape change, aggregation, and release reaction in response to synthetic TXA2 mimetic (STA2). When the platelet TXA2 receptor was examined with both a 125I-labeled derivative of a TXA2 receptor antagonist ([125I]-PTAOH) and [3H]-labeled TXA2 agonist ([3H]U-46619), the equilibrium dissociation rate constants (kd) and the maximal concentrations of binding sites (Bmax) of the platelets to both ligands were within normal ranges, suggesting that the binding capacity of their TXA2 receptor was normal. STA2 could not induce IP3 formation and intracellular Ca2+ mobilization, whereas these responses to thrombin were within normal ranges. GTPase activity was also decreased when the patient's platelet membrane was challenged with STA2. On the other hand, lysophosphatidylinositol formation, which is a direct indicator of phospholipase A2 (PLA2) activation, was found to be normal when the [3H]-inositol-labeled platelets were challenged with STA2. Thromboxane B2 (TXB2) was also produced in response to STA2. These results suggested that the abnormality in these platelets was impaired coupling between TXA2 receptor and phospholipase C (PLC) activation. Furthermore, it is also suggested that the activation of PLA2 and PLC are separable events in thromboxane-induced platelet activation.
High-density lipoproteins (HDLs) play a central role in transporting cholesterol from peripheral tissues to the liver for elimination from the body. Impairment of HDL-mediated cholesterol transport favors cholesterol deposition in the arterial wall and promotes development of arteriosclerosis. Tangier disease is a severe HDL deficiency syndrome characterized by the accumulation of cholesterol in tissue macrophages and prevalent atherosclerosis. A three-decade search for a culprit in Tangier disease led to the identification of mutations in a cell membrane protein called ABCA1, which mediates the secretion of excess cholesterol from cells into the HDL metabolic pathway. Because of its ability to deplete cells of cholesterol and to raise plasma HDL levels, ABCA1 has become a promising therapeutic target for preventing cardiovascular disease.
Inherited thrombocytopenias (IT) are a heterogeneous group of diseases caused by at least 26 different genes. At present, these genes account for approximately 50% of cases, suggesting that novel genes have yet to be identified for a comprehensive understanding of platelet biogenesis defects. This review provides an update of ITs focusing on the molecular basis and potential pathogenic mechanisms affecting megakaryopoiesis and platelet production.
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In this review, the authors summarize how platelets interact with subendothelium when the vessel wall is damaged or with intact endothelium in the inflammatory state. When subendothelium is exposed to rapidly flowing blood upon vessel damage, platelets adhere rapidly to the exposed surface, decelerate, and aggregate to arrest bleeding. Under high shear stress, such as is found in the microcirculation, the interaction between subendothelial von Willebrand factor (VWF) and its platelet receptor, glycoprotein (GP) Ib-IX-V, is required to slow down platelets and allow the platelet collagen receptors α2β1 and GP VI to bind to collagen. GP VI and α2β1 play important roles to activate platelets in the early stage and work with GP Ib-IX-V to fully activate platelets to form thrombi. GP Ib-IX-V and GP VI employ similar signaling pathways for platelet activation and the signals from both receptors are down-modulated by PECAM-1 (platelet–endothelial–cell adhesion molecule 1) to prevent unnecessary platelet activation under high shear. During inflammatory states, intact endothelial cells release VWF and P-selectin from their Weibel-Palade bodies. Both molecules are ligands for GP Ib-IX-V. The newly released VWF is larger and stickier than the form normally found in plasma and binds platelets spontaneously. Normally, VWF is processed by proteolysis by the plasma metalloprotease ADAMTS-13. Failure of this processing results in the microvascular thrombotic disorder thrombotic thrombocytopenic purpura. In this review, the authors also use available crystal structures of platelet receptors and ligands to explain the details of their interactions.
Defects in signal transduction mechanisms may underlie the impaired aggregation and secretion in patients with congenital platelet function defects (CPD). Both protein kinase C (PKC) induced pleckstrin phosphorylation and cytoplasmic Ca2+ mobilization play a major role in secretion. We postulated that combined platelet activation with a cell permeable direct PKC activator 1,2-dioctanoyl-sn-glycerol (DiC8) and ionophore A23187, which possibly bypass the steps involved in the intracellular synthesis of two major mediators (inositol trisphosphate, diacylglycerol), may induce normal dense granule secretion in patients with impaired receptor mediated secretion. We studied eight CPD patients with abnormal aggregation and secretion in response to several different surface receptor-mediated agonists despite the presence of normal dense granule contents. Receptor mediated Ca2+ mobilization and/or pleckstrin phosphorylation were abnormal in seven patients. Platelet activation with a combination of ADP (8 microM) with DiC8 (200 microM) or A23187 (10 microM) improved secretion in four patients. However, platelet activation with a combination of 200 microM DiC8 with 10 microM A23187, or 100 microM DiC8 with 5 microM A23187 induced normal secretion in platelet-rich plasma in all patients. These studies suggest that in such patients with CPD the ultimate process of exocytosis or secretion per se is intact and impaired secretion results from abnormalities in early signal transduction events, possibly upstream of diacylglycerol formation and calcium mobilization. Detailed studies are needed to delineate the specific abnormalities in these heterogenous patients with signal transduction defects.
CD69 is a signal transducing disulfide-linked homodimer functionally expressed on platelets, CD3bright thymocytes, and activated lymphocytes. In an attempt to investigate early molecular events in CD69-mediated cell activation we studied the relative contribution of phospholipase A2 (PLA2) and phosphatidylinositol-specific phospholipase C-dependent pathways during platelet activation induced by CD69 stimulation. Thromboxane A2 (TXA2) synthetase inhibitor and TXA2R inhibitor R68070 were able to inhibit platelet aggregation induced by CD69 stimulation, indicating that TXA2 was the main mediator of the response. CD69-induced arachidonic acid release and TXA2 production were essentially PLA2 dependent because they could be blocked by the PLA2 inhibitor quinacrine. Inositol 1,3,4-trisphosphate generation was clearly detectable after CD69 cross-linking, but it was completely abrogated by quinacrine and R68070 and therefore secondary to TXA2 release and TXA2R engagement. Finally, direct measurement of enzymatic activity in vitro using radiolabeled phospholipid vesicles showed that CD69 cross-linking resulted in PLA2-dependent arachidonic acid and lysophosphatidylcholine generation from phosphatidylcholine, which was sensitive to quinacrine but not to R68070. By contrast, CD69-induced 1,2-diacylglycerol release from phosphatidylinositol 4,5-bisphosphate was blocked by both inhibitors. These results indicate a preferential involvement of PLA2 in CD69-dependent signal transduction in platelets and provide evidence for the unique role of PLA2-mediated activation pathways in transmembrane receptor signaling.
We previously reported a patient with polycythemia vera whose platelets showed subnormal responses to thromboxane A2 (TXA2) (Thromb Haemostas 1987; 57: 158-64). The patient's platelets showed normal binding activity to TXA2 analogues but the generation of second messengers was defective. We further studied the mechanism of this dysfunction in this work. The coupling of TXA2 receptor with its relevant GTP binding protein was examined using STA2(a synthetic TXA2 mimetic)-induced augmentation of GTPase activity as its measure. Only subnormal increase in the GTPase activity of the patient's platelet membrane was found after STA2 stimulation. Down regulation of TXA2 receptor of the patient's platelet also showed abnormal behavior. These results suggested that the defective coupling of TXA2 receptor with GTP binding protein could be included as a cause of defective signal transduction in this patient's platelets. This defect might be due to the abnormality of TXA2 receptor itself if the receptor was down regulated by its phosphorylation through agonist-induced conformational change.
In 2 male patients (35 and 38 years) presenting with myocardial infarction an abnormal conversion of exogenous 14C-arachidonic acid by the patients' platelets, incubated in vitro, was observed. Neither patient's platelets showed evidence of a lipoxygenase pathway.
Platelet thromboxane formation from exogenous and endogenous substrate was high, while the platelet aggregation responses were normal. A myeloproliferative syndrome was excluded by bone marrow puncture. Similar defects have only been described so far in patients with myeloproliferative syndrome. This defect may be causative for the onset of clinical thrombotic events. It is speculative whether in vivo therapy with r-IFNα1c might be able to eradicate the pathological platelet clone.
Phosphoinositide/polyphosphoinositide (PI/PPI) metabolism, measured by the increase of 3H-phosphatidic acid (PA) and the decrease of 3H-phosphatidylinositol (PI) in 3H-arachidonate-labeled platelet suspensions, was assessed in five patients whose platelet functional defects included impaired initial rates of ADP, epinephrine and U44069 aggregation in platelet-rich plasma (PRP). In one patient, 3H-PA formation induced by collagen and thrombin was reduced or absent on two of three occasions, and the decrease in 3H-PI was reduced on one of these two occasions in response to collagen and A23187, and on all 3 occasions in response to thrombin. The variations in the formation of 3H-PA in this patient on different occasions broadly paralleled the variations in the initial rates of ADP and U44069 aggregation and in epinephrine aggregation seen in PRP. No such abnormalities of PI metabolism were found in four other patients with similar, but not identical, functional defects. These results suggest an impairment affecting metabolism of PI/PPI via the PI/PPI cycle in this patient's platelets. The association of abnormalities of PI metabolism with defects of initial platelet responses provides further support for a physiological role of phosphoinositide metabolism in the early activation mechanism of platelets.
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.
Aggregation responses to low concentrations of ADP, epinephrine, collagen and cationophore A23187 in platelets from two family members with marked bleeding tendencies were virtually absent, whereas shape change with ADP was normal. The contribution to factor X activation by collagen-treated platelets was markedly decreased. Glycoproteins IIb and III were also significantly reduced. The patients' platelets had normal stores of secretable constituents, but secretion of adenine nucleotides and acid hydrolases in response to low concentrations of thrombin and A23187 was drastically reduced compared to normal platelets; secretion of platelet factor 4 was normal. Agonist concentrations that normally produce maximal responses induced only partial aggregation and secretion in the patients' platelets. After prelabelling with [3H]arachidonate thrombin caused less changes in the [3H]phosphatidylinositol, [3H]phosphatidylcholine and free [3H]arachidonate in platelets from the patients than in platelets from normals. We conclude that the patients' platelets have an impairment in part of the signal processing mechanism that is common for all agonists and responses. This platelet abnormality, which has a superficial resemblance to thrombasthenia, represents a hitherto undescribed qualitative platelet disorder.
Haemorrhagic diatheses due to platelet function defects are a heterogenous and poorly understood group of conditions. We report the investigation of a female with a lifelong history of epistaxes, haemarthroses, menorrhagia and persistent iron-deficiency anaemia. Although platelet numbers and morphology were normal, platelet function was abnormal both in vivo and in vitro. Skin bleeding time was prolonged and aggregation thresholds in platelet-rich plasma to a variety of weak and strong agonists were increased. Platelet granule contents were normal and membrane glycoproteins GpIb and GpIIIa were present in normal amounts. Polyphosphoinositide metabolism and phosphatidic acid generation were diminished in thrombin-stimulated platelets, as was phosphorylation of the 47 kD substrate for protein kinase C and the 20 kD protein myosin light chain kinase, indicating impaired generation of the intracellular second messengers diacylglycerol and inositol trisphosphate due to diminished stimulated phospholipase C activity. Although intracellular free calcium, calmodulin activity and basal cAMP concentrations were normal, washed platelets showed increased cAMP accumulation following stimulation with prostaglandin E1 and forskolin. Platelet membrane lipid analysis revealed a reduction in plasmalogen phosphatidylethanolamine content. It is suggested that the membrane phospholipid abnormalities cause the abnormal platelet reactivity by interfering with signal transduction from platelet receptor, via intermediary G proteins, to phospholipase C and adenylate cylase. The bleeding tendency is likely to be a consequence of the altered stimulus-response coupling.
The rise in cytoplasmic ionized calcium concentration ([Ca2+]i) on platelet activation is a combination of Ca2+ release from internal stores and influx of extracellular Ca2+. To understand the underlying mechanisms, we studied internal release and influx of Ca2+ in platelets from four patients with impaired agonist-induced Ca2+ mobilization and abnormal platelet aggregation and secretion responses. In normal platelets, thrombin caused a dose-dependent increase in internal release and influx; aspirin inhibited the total rise in [Ca2+]i and influx but not internal release, indicating that internal release occurs independent of cyclooxygenase products. In the four patients, both internal release and influx of Ca2+ induced by thrombin and adenosine diphosphate were diminished; the defect was more striking at lower agonist concentrations. To determine whether the Ca2+ storage organelles of these platelets had a diminished responsiveness to inositol 1,4,5-triphosphate (IP3) we studied IP3 (0.05 to 5 mumol/L) induced Ca2+ release and found it to be normal in all patients. We conclude that the impaired Ca2+ mobilization in our patients is due to abnormalities in both internal release and influx and that it is unlikely to be due to impaired platelet responsiveness to IP3 or defective thromboxane production. The impaired Ca2+ mobilization may be due to defects in phospholipase C activation and IP3 production. These patients provide direct evidence that internal release and influx of Ca2+ on platelet activation are closely interrelated.
The Stormorken syndrome is a multifacetted syndrome including a bleeding tendency. No deviations were found in the coagulation- or fibrinolytic systems. Platelet number was low normal, and size abnormal, whereas EM findings were unremarkable. Survival time was half normal. Clot retraction was initially rapid, but clearly decreased, whereas prothrombin consumption was also initially rapid, but complete. Membrane GP's were normal, so was AA metabolism, PI-cycle, granule storage and secretion, and c-AMP function, whereas 5-HT uptake and storage was decreased. Optical platelet aggregation was low normal with all physiological agonists. The only clearly abnormal finding was that coagulant activity was present on non stimulated platelets at the same level as kaolin-stimulated normal platelets. This indicated a platelet abnormality which should lead to a thrombogenic, not to a haemorrhagic trait. This paradox may have its origin in rheology, because when challenged with in vivo shear rates in an ex vivo perfusion chamber, platelet cohesion was abnormally low. Further studies to better delineate the membrane abnormality are underway.
Five patients with mild bleeding tendencies characterized by defective thromboxane A2 (TXA2)-induced platelet aggregation are reported. The platelets of all the patients had the ability to bind exogenous TXA2. Bleeding time was markedly prolonged in one patient. In three of the five patients, synthetic TXA2 mimetic (STA2)-induced platelet responses, including IP3 formation, Ca2+ mobilization, phosphatidic acid formation and GTPase activities were selectively defective, suggesting impaired coupling between the TXA2 receptor and phospholipase C activation. However, in the remaining two patients, these responses were all within normal limits. This suggests that the defective site of this type of platelet disorder is heterogenous and that signaling mechanisms other than the TXA2 receptor-phospholipase C pathway are also involved in TXA2-induced platelet aggregation.
We describe an 11-year-old girl with a mild bleeding disorder since early childhood. The disorder was characterized by a prolonged bleeding time, and the patient's platelets showed defective aggregation responses to thromboxane A2 (TXA2) mimetic U46619 and arachidonic acid. In contrast, the platelets showed normal responses to thrombin and Ca ionophore A23187. When the platelet TXA2 receptor was examined with the [3H]-labeled TXA2 agonist U46619, the equilibrium dissociation rate constants (kd) and the maximal concentration of binding sites (Bmax) of the patient's platelets were within normal ranges. Normal GTPase activity was also induced in the patient's platelets by stimulation with U46619, however, inositol 1,4,5-triphosphate (IP3) formation was not induced by U46619. These results suggests that the patient's platelets had a defect in phospholipase C activation beyond TXA2 receptors.
Hemostasis is a result of interactions between fibrillar structures in the damaged vessel wall, soluble components in plasma, and cellular elements in blood represented mainly by platelets and platelet-derived material. During formation of a platelet plug at the damaged vessel wall, factors IXa and VIIIa form the "tenase" complex, leading to activation of factor X on the surface of activated platelets. Subsequently, factors Xa and Va form the "prothrombinase" complex, which catalyzes the formation of thrombin from prothrombin, leading to fibrin formation. An enhanced expression of negatively charged phosphatidylserine in the outer membrane leaflet resulting from a breakdown of the phospholipid asymmetry is essential for the formation of the procoagulant surface. An ATP-driven and inward-acting aminophospholipid "translocase" and a "floppase" counterbalancing this have been postulated to maintain the dynamic state of phospholipid asymmetry. A phospholipid-nonspecific "scramblase," believed to be responsible for the fast breakdown of the asymmetry during cell activation, has recently been isolated from erythrocytes, cloned, and characterized. An intracellular calcium-binding segment and one or more thioesterified fatty acids are probably of importance for calcium-induced activation of this transporter protein. Cytosolic calcium ions also activate the calcium-dependent protease calpain associated with shedding of microvesicles from the transformed platelet membrane. These are shed with a procoagulant surface and with surface-exposed P-selectin from the alpha-granules. Theoretically, therefore, microvesicles can be involved in both coagulation and inflammation. Scott syndrome is probably caused by a defect in the activation of an otherwise normal scramblase, resulting in a relatively severe bleeding tendency. In Stormorken syndrome, the patients demonstrate a spontaneous surface expression of aminophospholipids. Activated platelets and the presence of procoagulant microvesicles have been demonstrated in several clinical conditions, such as thrombotic and idiopathic thrombocytopenia, disseminated intravascular coagulation, and HIV-1 infection, and have been found to be associated with fibrin in thrombosis. Procoagulant microvesicles may also be formed from other cells as a result of apoptosis.
Inherited giant platelet disorders are extremely rare. The aim of this article is to review the clinical and laboratory features of this heterogeneous group and to arrive at a working classification. We conducted our literature search using the National Library of Medicine database. A total of 12 clinical entities were described. We classified them into 4 groups depending on the clinical and structural abnormalities. The pathophysiology of these disorders is largely unknown, and more research is needed, particularly in the light of recent advances in laboratory medicine. This review may provide a valuable reference for clinicians and may form a basis for future classification and research.
May-Hegglin anomaly (MHA) is an autosomal dominant macrothrombocytopenia of unclear pathogenesis characterized by thrombocytopenia, giant platelets and leukocyte inclusions. Studies have indicated that platelet structure and function are normal, suggesting a defect in megakaryocyte fragmentation. The disorder has been linked to chromosome 22q12-13. Here we screen a candidate gene in this region, encoding non-muscle myosin heavy chain A (MYH9), for mutations in ten families. In each family, we identified one of three sequence variants within either the -helical coiled coil or the tailpiece domain that co-segregated with disease status. The E1841K mutation was found in 5 families and occurs at a conserved site in the rod domain. This mutation was not found in 40 normal individuals. Four families had a nonsense mutation that resulted in truncation of most of the tailpiece. One family had a T1155I mutation present in an affected mother and daughter, but not in the mother's parents, thus representing a new mutation. Among the 30 affected individuals, 21 unaffected individuals and 13 spouses in the 10 families, there was correlation of a variant of MYH9 with the presence of MHA. The identification of MYH9 as the disease gene for MHA establishes the pathogenesis of the disorder, should provide further insight into the processes of normal platelet formation and may facilitate identification of the genetic basis of related disorders.
May-Hegglin anomaly (MHA) and Fechtner (FTNS) and Sebastian (SBS) syndromes are autosomal dominant platelet disorders that share macrothrombocytopenia and characteristic leukocyte inclusions. FTNS has the additional clinical features of nephritis, deafness, and cataracts. Previously, mutations in the nonmuscle myosin heavy chain 9 gene (MYH9), which encodes nonmuscle myosin heavy chain IIA (MYHIIA), were identified in all three disorders. The spectrum of mutations and the genotype-phenotype and structure-function relationships in a large cohort of affected individuals (n=27) has now been examined. Moreover, it is demonstrated that MYH9 mutations also result in two other FTNS-like macrothrombocytopenia syndromes: Epstein syndrome (EPS) and Alport syndrome with macrothrombocytopenia (APSM). In all five disorders, MYH9 mutations were identified in 20/27 (74%) affected individuals. Four mutations, R702C, D1424N, E1841K, and R1933X, were most frequent. R702C and R702H mutations were only associated with FTNS, EPS, or APSM, thus defining a region of MYHIIA critical in the combined pathogenesis of macrothrombocytopenia, nephritis, and deafness. The E1841K, D1424N, and R1933X coiled-coil domain mutations were common to both MHA and FTNS. Haplotype analysis using three novel microsatellite markers revealed that three E1841K carriers--one with MHA and two with FTNS--shared a common haplotype around the MYH9 gene, suggesting a common ancestor. The two new globular-head mutations, K371N and R702H, as well as the recently identified MYH9 mutation, R705H, which results in DFNA17, were modeled on the basis of X-ray crystallographic data. Altogether, our data suggest that MHA, SBS, FTNS, EPS, and APSM comprise a phenotypic spectrum of disorders, all caused by MYH9 mutations. On the basis of our genetic analyses, the name "MYHIIA syndrome" is proposed to encompass all of these disorders.
Chediak Higashi syndrome (CHS) is a rare, autosomal recessive disorder that affects multiple systems of the body. Patients with CHS exhibit hypopigmentation of the skin, eyes and hair, prolonged bleeding times, easy bruisability, recurrent infections, abnormal NK cell function and peripheral neuropathy. Morbidity results from patients succumbing to frequent bacterial infections or to an "accelerated phase" lymphoproliferation into the major organs of the body. Current treatment for the disorder is bone marrow transplant, which alleviates the immune problems and the accelerated phase, but does not inhibit the development of neurologic disorders that grow increasingly worse with age. There are several animal models of CHS, the beige mouse being the most characterized. Positional cloning and YAC complementation resulted in the identification of the Beige and CHS1/LYST genes. These genes encode a cytosolic protein of 430,000 Da. Sequence analysis identified three conserved regions in the protein: a HEAT repeat motif at the amino-terminus that contains several a helices, a BEACH domain containing the amino acid sequence WIDL, and a WD40 repeat motif, which is described as a protein-protein interaction domain. The presence of the BEACH and WD40 domains defines a family of genes that encode extremely large proteins.
Glanzmann Thrombasthenia, an exceptional inherited platelet disorder is characterized by a complete lack of platelet aggregation due to a defect in the alpha(IIb)beta(3) complex or to a qualitative abnormality of this complex. Advances in molecular biology have permitted to precise the molecular abnormality on alpha(IIb) or beta(3) genes responsible for the disease and have also contributed to a better knowledge of normal platelet physiology. Hemorrhages are the main clinical problem. Current principles of therapeutic management are proposed, with special reference to the risk of platelet alloimmunisation.
Bernard-Soulier Syndrome (BSS) is an autosomal recessive bleeding disorder due to quantitative or qualitative abnormalities in the glycoprotein (GP) Ib/IX/V complex, the platelet receptor for von Willebrand factor. BSS is characterized by giant platelets, thrombocytopenia, and prolonged bleeding time, and the hallmark of this disorder is the absence of ristocetin-induced platelet agglutination. In the last 10 years, the molecular and genetic bases of many GPIb/IX/V defects have been elucidated, providing a better understanding of primary hemostasis and structure-function relations of the complex. Thus far, more than 30 mutations of the GPIbalpha, GPIbbeta, or GPIX genes have been described in BSS. Recent studies also have shown that the phenotypes caused by mutations in the subunits of the GPIb/IX/V span a wide spectrum, from the normal phenotype, to isolated giant platelet disorders/macrothrombocytopenia, to full-blown BSS and platelet-type von Willebrand disease. Although recent progress in molecular biology has clarified the genotype-phenotype relationships of the GPIb/IX/V disorders, a close examination of platelet morphology on blood smears is still indispensable for a proper diagnosis. In this review, we summarize recent advances in the molecular basis of BSS with special emphasis on giant platelets and the genetic characteristics of Japanese BSS.
Glanzmann's thrombasthenia is an autosomal recessive disorder, rare in a global context, but a relatively more common platelet function defect in communities where consanguineous marriages are more frequent. On clinical grounds alone, it cannot be distinguished from other congenital platelet function defects. Epistaxis, gum bleeding, menorrhagia are the common clinical manifestations, whereas large muscle hematoma or hemarthrosis seldom occur in these patients. Essential diagnostic features are a normal platelet count and morphology, a greatly prolonged bleeding time, absence of platelet aggregation in response to ADP, collagen, epinephrine, thrombin and to all aggregating agents which ultimately depend on fibrinogen binding to platelets for this effect, flow cytometry, studies of GPIIb-IIIa receptors on the platelet membrane surface using monoclonal antibodies. The present review describes some of the uncommon features of the disorders and the currently available options which the treating physicians should be aware of during the management of these patients. Although by definition all patients with Glanzmann's thrombasthenia have a virtually complete failure of platelet aggregation, a number of variant forms of GT have been described in which the glycoproteins are present in normal or near normal amounts but are functionally defective. Understanding the pathophysiology of the disorder by the treating physicians is of utmost importance. Presence of high affinity platelet receptors resulting in thrombasthennia-like phenotype may require an antagonistic treatment atypical of classical GT management. It has now been established that different genetic mutations of either GPIIb or IIIa genes results in such a heterogeneity of thrombasthenia phenotype. Glanzmann's thrombasthenia is a paradigm for treating coronary artery disease patients with GPIIb-IIIa antibody and inhibitors. By using these medicines we create a temporary GT-like situation. Hence, understanding this disease is of utmost importance to the practicing cardiologist. As mutations for different variant forms of GT become known, our understanding of how GPIIb-IIIa molecules can be activated to act as a receptor for fibrinogen molecules will be increased. Such understanding undoubtedly will help us to devise better drugs with GPIIb-IIIa inhibitors. Molecular biology techniques have enabled us to equivocally detect heterozygote carriers who are clinically asymptomatic. However, there may be several laboratories in the developing world, which have no access to molecular biology techniques. Development of more robust techniques of quantitation of platelet receptors has enabled an accurate diagnosis of heterozygote carriers or an unborn fetus in the second trimester. The importance of the GPIIb-IIIa polymorphisms in carrier and prenatal diagnosis has not been properly studied. Nowadays the less direct method of PLA1 typing (determination of the levels of platelet antigen) of the foetal platelets as early as 16 weeks of intrauterine life can be used for prenatal diagnosis of GT.
The absence or deficiency of specific platelet glycoprotein receptors has a well-defined role in causing several rare bleeding disorders such as Bernard-Soulier syndrome or Glanzmann's thrombasthenia. Several new rare disorders caused by defects in other receptors or their signalling pathways have recently been described. Platelet receptors are also often targets for antibodies in pathological conditions. The roles of platelet receptors or their polymorphism variants in diseases such as cardiovascular disorders have started to be intensively investigated over the last 5 years. Many of these findings still remain controversial. Recent evidence points to a fundamental role for platelets and their receptors in the origins of atherosclerosis. Studies on the role of platelet receptors in diseases such as asthma, diabetes and HIV are still at an early stage.
Signaling events downstream from collagen receptors and G protein-coupled receptors are responsible for the initiation and extension of platelet plug formation. This creates the platelet plug and hopefully results in the cessation of bleeding. It is not, however, all that is required for hemostasis, and growing evidence is emerging that the perpetuation of a stable hemostatic plug requires additional intracellular signaling. At least part of this process is made possible by the persistent close contacts between platelets that can only occur after the onset of aggregation. This review discusses several examples of such signaling mechanisms that help to perpetuate the platelet plug in a contact-dependent manner, including outside-in signaling through integrins, signaling though Eph kinases and ephrins, and the role of CD40L.
Recent in vivo studies have highlighted the dynamic and complex nature of platelet thrombus growth and the requirement for multiple adhesive receptor-ligand interactions in this process. In particular, the importance of von Willebrand factor (VWF) in promoting both primary adhesion and aggregation under high shear conditions is now well established. In general, the efficiency with which platelets adhere and aggregate at sites of vessel wall injury is dependent on the synergistic action of various adhesive and soluble agonist receptors, with the contribution of each of the individual receptors dependent on the prevailing blood flow conditions. In this review, we will discuss the major platelet adhesive interactions regulating platelet thrombus formation under high shear, with specific focus on the VWF (GPIb and integrin alphaIIbbeta3) and collagen receptors (GPVI and integrin alpha2beta1). We will also discuss the signaling mechanisms utilized by these receptors to induce platelet activation with specific emphasis on the role of cytosolic calcium flux in regulating platelet adhesion dynamics. The role of soluble agonists in promoting thrombus growth will be highlighted and a model to explain the synergistic requirement for adhesive and soluble stimuli for efficient platelet aggregation will be discussed.
Insights into hemostasis and thrombosis have historically benefited from the astute diagnosis of human bleeding and thrombotic disorders followed by decades of careful biochemical characterization. This work has set the stage for the development of a number of mouse models of hemostasis and thrombosis generated by gene targeting strategies in the mouse genome. The utility of these models is the in depth analysis that can be performed on the precise molecular interactions that support hemostasis and thrombosis along with efficacy testing of various therapeutic strategies. Already the mouse has proven to be an excellent model of the processes that support hemostasis and thrombosis in the human vasculature. A brief summary of the salient phenotypes from knockout mice missing key platelet receptors is presented, including the glycoprotein (GP) Ib-IX-V and GP IIb/IIIa (alphaIIb/beta3) receptors; the collagen receptors, GP VI and alpha2/beta1; the protease activated receptors (PARs); and the purinergic receptors, P2Y(1) and P2Y(12). A few differences exist between mouse and human platelets and where appropriate those will be highlighted in this review. Concluding remarks focus on the importance of understanding the power and limitations of various in vitro, ex vivo and in vivo models currently being used and the impact of the mouse strain on the described platelet phenotype.
Glycoprotein (GP) VI is a platelet membrane protein with a molecular weight of 62 kDa that was identified as a physiological collagen receptor from studies of patients deficient in this protein. GPVI-deficient platelets lacked specifically collagen-induced aggregation and the ability to form thrombi on a collagen surface under flow conditions, suggesting that GPVI makes an indispensable contribution to collagen-induced platelet activation. On the platelet surface, GPVI is present as a complex with the Fc receptor (FcR) gamma-chain, probably composed of two GPVI molecules and one FcR gamma-chain dimer. GPVI must form such a dimeric complex to exhibit high affinity binding to collagen. The GPVI-induced activation mechanism is initiated by tyrosine phosphorylation of the immunoreceptor tyrosine-based activation motif (ITAM) of the FcR gamma-chain, and then this signal is transduced to many related proteins, mainly by tyrosine phosphorylation. GPVI is widely recognized as a requisite factor for the formation of platelet aggregates on a collagen surface under blood flow. However, individuals with GPVI-deficient or null platelets do not exhibit any strong bleeding tendency. Analyzing this apparent dichotomy should provide us with a more precise understanding of the mechanism of thrombus formation.
PH domains (pleckstrin homology domains) are the 11th most common domain in the human genome and are best known for their ability to target cellular membranes by binding specifically to phosphoinositides. Recent studies in yeast have shown that, in fact, this is a property of only a small fraction of the known PH domains. Most PH domains are not capable of independent membrane targeting, and those capable of doing so (approx. 33%) appear, most often, to require both phosphoinositide and non-phosphoinositide determinants for their subcellular localization. Several recent studies have suggested that small GTPases such as ARF family proteins play a role in defining PH domain localization. Some others have described a signalling role for PH domains in regulating small GTPases, although phosphoinositides may also play a role. These findings herald a change in our perspective of PH domain function, which will be significantly more diverse than previously supposed.
The last several years have seen an abundance of studies of genetic risk factors for vascular disease, and platelet glycoprotein (GP) polymorphisms have been a primary focus of this area of research. This article reviews GP receptor polymorphisms, particularly those on GPIa-IIa (integrin alpha2beta1), GPIb-IX-V, GPIIb-IIIa (integrin alpha(IIb)beta3), and GPVI, and summarizes clinical and functional studies that have attempted to clarify their roles in human disease. Our focus is on recent work relevant to thrombotic and hemostatic processes and advances in pharmacogenetics. We consider issues affecting our ability to derive firm conclusions from these studies, and discuss future directions in this rapidly evolving area.
Platelet dense granules form using mechanisms shared by melanosomes in melanocytes and by subsets of lysosomes in more generalized cells. Consequently, disorders of platelet dense granules can reveal how organelles form and move within cells. Models for the study of new vesicle formation include isolated delta-storage pool deficiency, combined alphadelta-storage pool deficiency, Hermansky-Pudlak syndrome (HPS), Chediak-Higashi syndrome, Griscelli syndrome, thrombocytopenia absent radii syndrome, and Wiskott-Aldrich syndrome. The molecular bases of dense granule deficiency are known for the seven subtypes of HPS, as well as for Chediak-Higashi syndrome, Griscelli syndrome, and Wiskott-Aldrich syndrome. The gene products involved in these disorders help elucidate the generalized process of the formation of vesicles from extant membranes such as the Golgi.
Hermansky-Pudlak Syndrome (HPS) is a genetically heterogeneous disorder in which mutations in one of several genes interrupts biogenesis of melanosomes, platelet dense bodies, and lysosomes. Affected patients have oculocutaneous albinism, a bleeding diathesis, and sometimes develop granulomatous colitis or pulmonary fibrosis. In order to assess the role of HPS genes in melanosome biogenesis, melanocytes cultured from patients with HPS subtypes 1, 2, or 3 were assessed for the localization of various melanocyte proteins. Tyrosinase, Tyrp1, and Dct/Tyrp2 were atypically and distinctly expressed in HPS-1 and HPS-3 melanocytes, whereas only tyrosinase showed an atypical distribution in HPS-2 melanocytes. The HPS1 and AP3B1 (i.e., HPS-2) gene products showed no expression in HPS-1 and HPS-2 melanocytes, respectively, whereas HPS-3 melanocytes exhibited normal expression for both proteins. In normal human melanocytes, the HPS1 protein was expressed as an approximately 80 kDa molecule with both granular and reticular intracellular profiles. In HPS-1, lysosome associated membrane protein 1 (LAMP1), and LAMP3 were localized to abnormal large granules; in HPS-2, all LAMPs exhibited a normal granular expression; and in HPS-3, LAMP1, and LAMP3 exhibited a distinct less granular and more floccular pattern. In contrast, the expressions of Rab 27, transferrin, and cKit were unaffected in all three HPS genotypes. These data demonstrate that the three initially identified subtypes of human HPS exhibit distinct defects in the trafficking of various melanocyte-specific proteins.
The tetraspanins are a superfamily of membrane glycoproteins which facilitate the interaction of membrane and intracellular signalling molecules (e.g., integrins, pro-growth factors and their receptors, protein kinase-C) in the formation of membrane signalling microdomains (sometimes referred to as the tetraspanin web). A proximal localisation/association of tetraspanins with Fc receptors (FcR) has been implied by the repeated rediscoveries of tetraspanins as the targets of antibodies which activate platelets and other blood cells through co-ligation of FcR. Direct evidence of tetraspanin-FcR interactions has come from immunoprecipitation and co-immunofluorescence studies. The functional effects of this interaction remain unclear, but tetraspanins have been identified as negative regulators of FcR signalling independently of co-ligation, indicating potential roles in modulating FcR function in co-ordination with the activity of other signalling/adhesion molecules in the tetraspanin web. Given their capacity to influence FcR signalling, tetraspanins could provide specific therapeutic targets for immune disorders including rheumatoid arthritis, asthma and allergies.
Platelet factor 4 (PF4) is a platelet alpha-granule protein sequenced over 25 years ago that is a founding member of the C-X-C chemokine family, yet its physiologic function has yet to be definitively established. Initial investigations focused on possible procoagulant roles for PF4 in platelet function and plasmatic coagulation. Subsequent in vitro studies have, however, described a puzzling array of other apparently unrelated biologic functions, including inhibition of angiogenesis and hematopoiesis, promotion of neutrophil adhesion, and activation, enhancement of oxy-LDL binding to the LDL receptor and stimulation of anti-coagulant activated protein C generation by the thrombomodulin/protein C system. Preliminary studies with a just-described PF4 knockout mouse line support a role for PF4 in platelet-dependent thrombosis in vivo.