Shortened Platelet Survival Research Articles

Complement activation in patients with immune thrombocytopenic purpura according to phases of disease course.

Immune thrombocytopenic purpura (ITP) is an autoimmune thrombocytopenia with shortened platelet survival and relative bone marrow failure. The pathogenesis involves antibody production, cytokine release, T cell impairment, complement activation and clearance of platelets. We measured plasma levels of C3, C4, C1q and sC5b-9 in 80ITP patients in acute phase, 50ITP patients in complete (CR) or partial (PR) remission and 50age- and sex-matched healthy volunteers. Statistical analyses showed that acute ITP patients had higher plasma levels of sC5b-9 and C1q than CR or PR patients (median=sC5b-9: 200versus98mg/dl, P-value<0·001) (median C1q=2·11 versus 1·00 mg/dl, P-value<0·001). CR and PR ITP patients had sC5b-9 and C1q plasma levels comparable to those observed in healthy volunteers. There was a significant correlation between sC5b-9 and C1q plasma levels (Spearman's rho correlation index on 130 ITP patients equal to 0·58, P-value<0·001). We also found that sC5b-9 plasma level is inversely correlated with the number of platelets. Furthermore, we divided acute ITP patients into subjects with detectable (24 of 80, 30%) or undetectable (56 of 80, 70%) anti-platelet antibodies; patients with detectable anti-platelet antibodies have significantly higher plasma levels of C1q and sC5b-9. This research will potentially offer novel therapeutic strategies in light of new drugs affecting complement activation for monitoring therapy response.

PS1491 PLATELET KINETICS IN PATIENTS WITH CHRONIC IMMUNE THROMBOCYTOPENIA TREATED WITH THROMBOPOIETIN RECEPTOR AGONISTS

Background:Chronic immune thrombocytopenia (ITP) is characterised by antibody‐mediated platelet destruction, shortened platelet survival (PS) and impaired platelets production. Platelet kinetic study (PKS) assesses in vivo the mean PS, platelet production and the platelet sequestration site. Thrombopoietin receptor agonists (TPO‐RA) increase platelets counts (PC) in majority of chronic ITP patients. Moreover, some patients could retain stabile response after TPO‐RA discontinuation, which is attributed to the immunomodulatory effects of the drugs. Addressing this issue only a few studies have been done assessing the effect of TPO‐RA on PKS parameters.Aims:To evaluate the effects of TPO‐RA on PKS parameters.Methods:Ten patients with chronic ITP, median age 55.3 years (range 12–72), gender (f: n = 7, m: n = 3), splenectomised 6/10, were treated with TPO‐RA (eltrombopag, n = 7, romiplostim, n = 3). Response was assessed according to the criteria of the International Working Group (IWG) on ITP. Autologous 111Induim‐oxinate‐labeled PKS were performed in all patients before and 5 months after TPO‐RA therapy initiation.Results:A good treatment response was achieved in 8/10 of patients (eltrombopag, n = 6, romiplostim, n = 2). Two refractory patients were treated with TPO‐RA and low dose prednisolone. Baseline median parameters were as follows: PC 6.5x109/L (range 1–17), PS 0.5 days (range 0.1–1.6, normal: 7–10), platelet production ratio 0.65 (range 0.01–3.2, normal 1 ± 0.2), platelet turnover 30875 Plt/μL/day (range 1731–103500). The post−treatment PKS showed significantly higher median PC of 62.5x109/L (range 5–501) (p = 0.013) and median platelet production ratio of 1.8 (range 0.5–37.9) (p = 0.05). However, median PS of 0.7 days (range 0.1–2.9) (p = 0.09), as well as platelet turnover of 58471.5 Plt/μL/day (range 2800–462236) (p = 0.086) and platelet destruction site remained unaltered. It is of note that two refractory patients (N08 and N010 in table) increased their platelet production (platelet production ratio and platelet turnover), while their PS remained reduced. It is of note that there was no clear correlation between PC and PS. Nevertheless, PC correlated only with platelet turnover prior (p = 0.006) and after TPO‐RA therapy (p = 0.029).Summary/Conclusion:Our prospective study in which PKS was performed initially and during TPO‐RA therapy suggests that TPO‐RA could increase PC by augmented production. Further studies on larger cohorts are needed to confirm this idea.image

SAT-605 Immune Thrombocytopenic Purpura in Graves' disease

Background: Immune thrombocytopenic purpura (ITP) in association with autoimmune thyroid diseases is a well-known entity. We report a case of Graves’ disease patient with coexisting ITP, where the control of ITP is in close relationship to control with Graves’ disease. Case Presentation: A 17-year-old female presented in 10/2014 with hyperthyroid symptoms for 4-5 months. Thyroid function tests (TFT’s) showed TSH 0.011 (0.45-4.5 mcIU/ml) and Total T4 (TT4) - 34.3 (4.5-12 ug/dl). Thyroid ultrasound showed thyromegaly without any discrete nodules. Thyroid uptake scan showed a homogeneous increased radiotracer uptake of 65% at 24 hours consistent with Graves’ disease and she was started on Methimazole (MMI) 20 mg PO daily. Over time, her hyperthyroidism remained well controlled on MMI 5 mg q other day, hence it was discontinued in 5/2016. She presented with multiple skin bruises in 12/2016 and her platelet count was noted to be 19 (150-400x103/ml). TFT’s remained normal. Her presumptive diagnosis was ITP given another autoimmune condition - Graves’ disease and was started on Prednisone 80 mg daily. She followed with hematology and her platelet count was monitored regularly and prednisone dose was adjusted. In 7/2017 her platelet count dropped to 64 on prednisone 5 mg PO daily. Repeat TFT’s showed TSH 0.01 and free T4 3.31. MMI was resumed at 20 mg PO daily. Labs three months later show TSH 0.024, free T4 0.86, TT3 102 and platelets 186. Given the recurrence of Graves’ disease she underwent radioactive iodine (RAI) I131 therapy in 1/2018. Post RAI she required levothyroxine 50 mcg daily briefly. In 5/2018 her platelet count dropped again to 59 on prednisone. TFT’s showed TSH of 0.014, free T4 1.42 and TT3 117. Graves’ disease relapsed despite RAI therapy and MMI was started at 5 mg PO daily. Her thyroid function remains well controlled on MMI 5 mg PO daily. Her platelet count also returned to normal and remains stable on Prednisone 5 mg PO daily. Discussion: There are several case reports of Graves’ disease and ITP in the literature. Several mechanisms by which thrombocytopenia occurs have been postulated. Accelerated platelet turnover by an activated reticuloendothelial system, shortened platelet survival and consequently reduced platelet counts can occur in patients with hyperthyroidism. Platelet survival return to normal in patients maintained in euthyroid state for more than six months. Genetic predisposition to both Graves’ disease and ITP appears to be related to HLA B8 and DR3 haplotypes. In our patient, each time she had a low platelet count on prednisone therapy, thyroid function tests were suggestive of relapse of Graves’ disease. Treatment of Graves’ disease has resulted in improved response of ITP to glucocorticoid therapy and normalization of platelet count. In patients with ITP on glucocorticoid therapy, unexplained drop in platelet count should prompt checking for thyroid function abnormalities.

Aspirin-Responsive, Migraine-Like Transient Cerebral and Ocular Ischemic Attacks and Erythromelalgia in JAK2V617F-Positive Essential Thrombocythemia and Polycythemia Vera

Migraine-like cerebral transient ischemic attacks (MIAs) and ocular ischemic manifestations were the main presenting features in 10 JAK2^V617F-positive patients studied, with essential thrombocythemia (ET) in 6 and polycythemia vera (PV) in 4. Symptoms varied and included cerebral ischemic attacks, mental concentration disturbances followed by throbbing headaches, nausea, vomiting, syncope or even seizures. MIAs were frequently preceded or followed by ocular ischemic events of blurred vision, scotomas, transient flashing of the eyes, and sudden transient partial blindness preceded or followed erythromelalgia in the toes or fingers. The time lapse between the first symptoms of aspirin-responsive MIAs and the diagnosis of ET in 5 patients ranged from 4 to 12 years. At the time of erythromelalgia and MIAs, shortened platelet survival, an increase in the levels of the platelet activation markers β-thromboglobulin and platelet factor 4 and also in urinary thromboxane B2 were clearly indicative of the spontaneous in vivo platelet activation of constitutively JAK2^V617F-activated thrombocythemic platelets. Aspirin relieves the peripheral, cerebral and ocular ischemic disturbances by irreversible inhibition of platelet cyclo-oxygenase (COX-1) activity and aggregation ex vivo. Vitamin K antagonist, dipyridamole, ticlopidine, sulfinpyrazone and sodium salicylate have no effect on platelet COX-1 activity and are ineffective in the treatment of thrombocythemia-specific manifestations of erythromelalgia and atypical MIAs. If not treated with aspirin, ET and PV patients are at a high risk of major arterial thrombosis including stroke, myocardial infarction and digital gangrene.

Managing the platelet refractory patient

Platelet refractoriness is the repeated failure to obtain satisfactory responses to platelet transfusions. It is mainly due to shortened platelet survival associated with non‐immune clinical factors, such as infection and its treatment with antibiotics and antifungal drugs, disseminated intravascular coagulation (DIC) and splenomegaly. The main immune cause is HLA alloimmunization which occurs predominantly in females with a history of pregnancy. The incidence of alloimmune platelet refractoriness due to HLA antibodies has declined due to leucocyte reduction in blood components and more aggressive treatment for patients with haematological malignancies and other cancers. Responses to platelet transfusions should be carefully monitored. If platelet refractoriness occurs, a clinical assessment should be made for clinical factors associated with non‐immune platelet consumption. If there are no obvious clinical factors present, an immune mechanism should be suspected and testing for HLA antibodies conducted. If HLA antibodies are detected, HLA‐matched platelet transfusions should be used. There are a number of methods including computer algorithms to determine HLA compatibility and acceptable HLA mismatches. HLA‐matched platelet transfusions are not indicated when serological testing has failed to detect HLA antibodies; further consideration should be given to the presence of non‐immune clinical factors and testing for HPA antibodies. If there are improved responses to HLA‐matched platelet transfusions, they should be continued. If there are poor responses to HLA‐matched platelet transfusions, the reasons should be sought including HLA incompatibility which is most likely to occur in patients with unusual HLA types with few well‐matched donors, non‐immune platelet consumption, and HPA and ABO incompatibility. Further serological investigations including testing for HPA antibodies and/or platelet cross‐matching may be used to differentiate between these possibilities. The management of patients with HLA and/or HPA alloimmunization and no compatible donors may be very difficult. The management of patients with non‐immune platelet consumption is similarly problematic.

Flow cytometric assessment of canine erythrocytes and platelets for dog erythrocyte antigen 1.1

In human medicine, transfusion of ABO-mismatched platelets has been associated with shortened platelet survival and refractoriness to platelet transfusion because of expression of certain blood group antigens on platelets. It remains unknown if canine platelets express dog erythrocyte antigens (DEAs). The aim of this study was to develop a flow cytometric assay for DEA 1.1 and determine whether DEA 1.1 is present on canine platelets. Blood was collected from 172 clinically healthy dogs. Platelets and erythrocytes from each dog were tested for DEA 1.1 by flow cytometry using anti-DEA 1.1 blood-typing sera. Erythrocytes from each dog were also assessed for DEA 1.1 using a standard tube-typing test (T1) and using a second tube method (T2), if the flow cytometric and T1 results differed. Using flow cytometry, DEA 1.1 was detected on erythrocytes of all 110 dogs shown by T1 or T2 testing to be DEA 1.1-positive. Initial results of the T1 test had a diagnostic accuracy of 93% (160 correct/172 tests). The frequency of erythrocyte DEA 1.1 positivity in previously untyped dogs (n = 118) was 56%. DEA 1.1 expression was not detected on platelets from DEA 1.1-positive dogs. Flow cytometry was a reliable method for detection of DEA 1.1 on canine erythrocytes. The absence of DEA 1.1 on platelets from DEA 1.1-positive dogs suggests that their platelets do not express DEA 1.1 and will not induce production of anti-DEA 1.1 antibodies that might lead to platelet refractoriness or reactions to a subsequent transfusion of DEA 1.1-positive erythrocytes.

Aspirin

> Among the many useful discoveries which this age has made, there are very few which better deserve the attention of the public that what I am going to lay before your Lordship. > > —Reverend Edward Stone –Chipping-Norton, Oxfordshire –April 25, 1763 These prophetic words, written by Reverend Edward Stone in a 1763 letter to George Parker, the second Earl of Macclesfield, describe the results of the first clinical trial recorded in medical history.1 Stone's report on the rediscovery of the medicinal value of willow bark among subjects suffering from malarial symptoms is considered a significant milestone in the development of aspirin. Although society now takes many of its beneficial effects for granted, aspirin did not suddenly appear for medicinal use after Reverend Stone's discovery. Instead, its tumultuous journey was fueled by individual scientific curiosity, accidental discoveries, and intense business rivalry.1 No other drug is used by a greater number of people worldwide than aspirin, the benefits of which span centuries, beginning with the very first uses of willow bark by Egyptian physicians (Figure 1). Aspirin single-handedly transformed a coal-dye company into a pharmaceutical giant and has emerged as a cornerstone in the present-day therapies available for treating cardiovascular disease (CVD), pain, and inflammation. This article discusses the sentinel historical aspects of the discovery and clinical cardiovascular developments of aspirin, as well as its contemporary use in today's medical arena. Figure 1. Timeline of historical events in the development of aspirin. ### Historical Developments of Salicylates On January 20, 1862, Edwin Smith made one of the most historically important purchases of his life. Well-regarded among his peers for his keen scholarship and intricate knowledge of Egyptology, Smith purchased, for £12, 2 worn papyrus scrolls in a local Luxor street market1 that later turned out to be a formative medical textbook unlocking ancient Egyptian's practice of medicine. Although authorless, the Ebers Papyrus is 110 pages …

Mechanisms of resveratrol-induced platelet apoptosis

Apoptotic events have recently been found to occur in platelets, which are anuclear. Resveratrol is present in red wine and has various biological activities, including inhibition of platelet aggregation. Although considerable evidence is available as to the induction of tumour cell apoptosis by resveratrol, resveratrol's effects on platelet apoptosis have not yet been investigated. In the present study, we demonstrate that resveratrol also markedly stimulates apoptosis in washed human platelets. Resveratrol (5-25 microM) completely inhibited platelet aggregation stimulated by collagen. Furthermore, resveratrol time- and concentration-dependently stimulated dissipation of the mitochondrial membrane potential (DeltaPsim), activation of caspases-9, -3, and -8, gelsolin and actin cleavage, Bid cleavage into truncated Bid, Bax translocation, cytochrome c release, and phosphatidylserine exposure but not P-selectin expression in washed human platelets. The presence of z-IETD-fmk, a caspase-8 inhibitor, markedly reversed tBid formation and caspase activation and partially reversed the dissipation of platelet DeltaPsim stimulated by resveratrol. In addition, resveratrol also directly evoked dissipation of DeltaPsim and release of cytochrome c from isolated mitochondria. Furthermore, resveratrol shortened platelet survival or enhanced platelet clearance in an in vivo study. This study demonstrates for the first time that resveratrol simultaneously inhibits platelet aggregation and stimulates platelet apoptosis. Stimulation of platelet apoptosis by resveratrol may represent the increased therapeutic potential for patients suffering from thrombotic conditions or thrombocytosis to promote platelet destruction and thus prevent pathological clotting. Furthermore, this study also provides a novel conception that rigorous surveillance of platelet numbers may be important during resveratrol treatment in the clinic.

Thrombopoietin (TPO) Levels in Inflammatory Disorders with or without Reactive Thrombocytosis.

TPO, the primary regulator of megakaryopoiesis, is produced at a constant rate and is removed from the circulation by adsorption to specific receptors on platelet and megakaryocyte surfaces. An inverse relationship between platelet count and TPO therefore exists in healthy subjects. However, a few studies in patients with reactive thrombocytosis identified levels of the hormone higher than expected, and suggested that TPO behaves as an acute-phase protein and was responsible for increased platelet count. At the opposite, other studies did not find any significant rise of the hormone in patients who similarly developed reactive thrombocytosis.To gain further information on this topic, we compared TPO levels and platelet counts in two series of hospitalized patients: one (53 patients aged 73 ± 17 years) with strong elevation of both erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP), and the other (28 age-matched control patients) with normal values. All subjects had been admitted to hospital for acute illnesses: infections, cancer, heart failure or ischemia, stroke, deep vein thrombosis or pancreatitis. Within the group of subjects with high ESR and CRP, 38 had normal platelet counts, while 15 had thrombocytosis at the admittance (n=9) or developed it during their stay (n=6). No thrombocytosis was observed in control patients. Patients with high acute phase indexes had significantly higher TPO levels (R&D Systems, Minneapolis, USA) and platelet counts than control patients (TPO 189.55pg/ml ± 139 vs 62.04pg/ml ± 83, p<0.0001; platelets 310×109/L ± 98 vs 242×109/L ± 67, p=0.0016). Since similar TPO and platelet values were found in patients grouped according to their disorders, we assumed that the acute phase, but not the basic illness, was responsible for the increase of TPO and platelets. We identified strong, positive correlations between ESRc (ESR corrected for Hct) and CRP (r=0.809, p<0.0001), but only weak correlations between TPO and CRP (r=0.584, p<0.0001). Interestingly, no significant relationship between platelet counts and TPO levels (r=0.180, p=NS) was find. When we grouped patients with acute-phase reaction according to absence (platelets 280×109/L ± 61) or presence (platelets 516×109/L ± 105) of thrombocytosis, we found similar TPO values (184pg/ml ± 146 vs 203.4pg/ml ± 126, p=NS). By measuring plasma glycocalicin (GC, Takara Bio Inc. Shiga, Japan) and estimating the glycocalicin index (GCI, by normalizing GC levels for the individual platelet count), which reflects platelet turnover, we excluded that shortened platelet survival masked increased platelet production in subjects with inflammatory conditions and normal platelet count. In fact, identical values of GCI were found in patients with normal platelet count or thrombocytosis (2.434 ± 1.4 vs 2.496 ± 2.5). Finally, no correlation was detected between TPO and GCI (r=0.003, p=NS). Conversely, a strong positive correlation between IL-6 (R&D Systems) and CRP (r=0.692, p<0.0001), and a trend towards correlation between platelet count and IL-6 (r=0.493, p<0.0001) and between TPO and IL-6 (r=0.484, p<0.0001) have been identified. All together our results confirm that TPO acts as an acute phase protein but exclude the possibility that it is uniquely responsible for thrombocytosis of inflammatory disorders, which might recognize in IL-6 a credible candidate as a cooperating factor.

The Paradox of Platelet Activation and Impaired Function: Platelet-von Willebrand Factor Interactions, and the Etiology of Thrombotic and Hemorrhagic Manifestations in Essential Thrombocythemia and Polycythemia Vera

Patients with essential thrombocythemia (ET) and polycythemia vera (PV), complicated by microvascular ischemic or thrombotic events, have shortened platelet survival, increased beta-thromboglobulin, platelet factor 4, and thrombomodulin levels, and increased urinary thromboxane B2 excretion. These are all reversible by inhibition of platelet cyclooxygenase 1 with aspirin, and are therefore indicative of platelet activation and platelet-mediated thrombotic processes. The thrombotic tendency persists as long as platelet counts are above the upper limit of normal (400 x 10 (9)/L). Despite strong evidence of in vivo platelet activation, the ex vivo platelet function tests are impaired. Platelet dysfunction in ET and PV typically is characterized by a missing second-wave adrenaline aggregation, an increased adenosine diphosphate aggregation threshold, and reduced secretion products, but a normal arachidonic acid or collagen-induced aggregation. The proposed concept is that platelets in thrombocythemia (ET and PV) are hypersensitive. Due to the existing high shear stress in the microvasculature (end-arterial circulation), platelets spontaneously activate, secrete their products, form aggregates mediated by von Willebrand factor (vWF) that transiently plug the microcirculation, deaggregate, and then recirculate as exhausted defective platelets with secondary storage pool disease on ex vivo analysis. At increasing platelet counts from below to above 1000 x 10 (9)/L, the thrombotic condition changes into an overt spontaneous bleeding tendency as a result of a functional vWF deficiency that is caused by proteolysis of large vWF multimers. This is consistent with acquired type 2 von Willebrand syndrome (AvWS). AvWS is reversible by reduction of the platelet count to normal. The acquired JAK2 V617F gain of function mutation is the cause of trilinear myeloproliferative disease with the sequential occurrence of ET and PV. Heterozygous JAK2 V617F mutation with slightly increased kinase activity is enough for the induction of spontaneous megakaryopoiesis and erythropoiesis, and an increase of hypersensitive platelets is the cause of aspirin-sensitive, platelet-mediated microvascular ischemic and thrombotic complications in ET and early PV mimicking ET. Homozygous JAK2 mutation with pronounced increase of kinase activity is associated with pronounced trilinear megakaryocyte, erythroid, and granulocytic myeloproliferation, with the most frequent clinical picture of classical PV complicated by major thrombosis, in addition to the platelet-mediated microvascular thrombotic syndrome of thrombocythemia.

Clinical and Laboratory Features, Pathobiology of Platelet-Mediated Thrombosis and Bleeding Complications, and the Molecular Etiology of Essential Thrombocythemia and Polycythemia Vera: Therapeutic Implications

Microvascular disturbances in essential thrombocythemia (ET) and polycythemia vera (PV), including erythromelalgia, and atypical and typical transient cerebral, ocular, and coronary ischemic attacks, are caused by platelet-mediated transient and occlusive thrombosis in the end-arterial circulation. ET patients with microvascular disturbances have shortened platelet survival, increased beta-thromboglobulin (beta-TG), platelet factor 4 (PF4), and thrombomodulin (TM) levels, and increased urinary thromboxane B2 (TXB2) excretion, indicating platelet-mediated thrombotic processes. Inhibition of platelet cyclooxygenase-1 by aspirin is followed by relief of microvascular disturbances; correction of shortened platelet survival; correction of increased plasma beta-TG, PF4, and TM levels; and correction of increased TXB2 excretion to normal. In PV associated with thrombocythemia, increased hematocrit and whole blood viscosity aggravate the platelet-mediated microvascular syndrome of thrombocythemia to produce major arterial and venous thrombotic complications. Correction of hematocrit to normal by phlebotomy will reduce the major arterial and venous thrombotic complications, but fails to prevent the platelet-mediated microvascular circulation disturbances in PV patients because thrombocythemia persists. Complete relief and prevention of microvascular and major thrombosis in ET and PV patients, in addition to phlebotomy, are obtained by treatment with aspirin and not with coumarin. The discovery of JAK2 V617F gain of function mutation in patients with myeloproliferative disorders (MPDs) expands our insights into the molecular etiology and biological features of ET, PV, and chronic idiopathic myelofibrosis (CIMF). The current concept is that heterozygous JAK2 V617F mutation with increased kinase activity is enough for megakaryocyte proliferation and increased hypersensitive platelets with no or slightly increased erythropoiesis in ET and in early PV mimicking ET. Homozygous JAK2 mutation with pronounced kinase activity is associated with trilinear megakaryocyte, erythroid, and granulocytic myeloproliferation, myeloid metaplasia, and secondary myelofibrosis (MF), with the most frequent clinical picture of classical PV complicated by major thrombosis in addition to the platelet-mediated microvascular thrombotic syndrome of thrombocythemia. The positive predictive value of a JAK2 V617F polymerase chain reaction test for the diagnosis of MPDs is high (near to 100%), but only half of ET and MF (sensitivity 50%) and the majority of PV (sensitivity 85 to 97%) are JAK2 V617F positive. Bone marrow histopathology, when used in combination with specific markers such as serum erythropoietin, PRV-1, endogenous erythroid colony formation, peripheral blood parameters and red cell mass, has a high sensitivity and specificity (near 100%) to detect the early and overt stages of the MPDs and to differentiate between ET, PV, and CIMF in both JAK2 V617F-positive and -negative MPDs.

Platelet-mediated erythromelalgic, cerebral, ocular and coronary microvascular ischemic and thrombotic manifestations in patients with essential thrombocythemia and polycythemia vera: A distinct aspirin-responsive and coumadin-resistant arterial thrombophilia

Microvascular circulation disturbances including erythromelalgia, its microvascular ischemic complications, and migraine-like atypical or typical transient ischemic cerebral, ocular, and coronary ischemic attacks are specific clinical manifestations in patients with essential thrombocythemia (ET), and polycythemia vera (PV) associated with thrombocythemia. Thrombocythemia (ET and PV) patients with microvascular disturbances have shortened platelet survival, increased β-thromboglobulin (β-tg), platelet factor 4 (PF4), and thrombomoduline (TM) levels, and increased urinary thromboxane B2 (TxB2) excretion indicating platelet-mediated processes in vivo. Inhibition of platelet cyclooxygenase (COX 1) by aspirin is followed by relief of microvascular disturbances, correction of shortened platelet survival, and return of plasma levels of β-tg, PF4, TM levels and TxB2 excretion to normal. The transient ischemic attacks and thrombotic complications in thrombocythemia are very likely caused by hypersensitive platelets produced by spontaneously proliferating enlarged megakaryocytes in the bone marrow of ET and PV patients. In contrast to normal platelets in healthy individuals the circulating hypersensitive thrombocythemic platelets spontaneously activate and secrete their products, thus forming aggregates that transiently plug the microcirculation, or result in occlusive platelet thrombi in arterioles or small arteries. Clear evidence is presented that the microvascular transient ischemic and occlusive thrombotic complications in thrombocythemia patients are relieved by treatment with aspirin and by reduction of platelet counts to normal (<400 × 109/l), but not by coumadin. In patients with thrombocythemia associated with PV, increased hematocrit and whole blood viscosity aggravate the platelet-mediated microvascular ischemic and thrombotic syndrome of thrombocythemia to major arterial and venous thrombotic complications. Correction of hematocrit and blood viscosity by phlebotomy significantly reduces the major arterial and venous thrombotic complications, but fails to prevent the platelet-mediated microvascular circulation disturbances in PV patients because thrombocythemia persists. Complete relief and prevention of microvascular and major thrombosis in PV patients are obtained by treatment with low-dose aspirin on top of phlebotomy or by treatment with the platelet lowering agents, anagrelide, interferon or hydroxyurea.

SCN9A Mutations Define Primary Erythermalgia as a Neuropathic Disorder of Voltage Gated Sodium Channels

Primary erythermalgia is a rare disorder characterized by recurrent attacks of red, warm and painful hands, and/or feet. We previously localized the gene for primary erythermalgia to a 7.94 cM region on chromosome 2q. Recently, Yang et al identified two missense mutations of the sodium channel alpha subunit SCN9A in patients with erythermalgia. The presence of voltage-gated sodium channels in sensory neurons is thought to play a crucial role in several chronic painful neuropathies. We examined four different families and two sporadic cases and detected missense sequence variants in SCN9A to be present in primary erythermalgia patients. A total of five of six mutations were located in highly conserved regions. One family with autosomal dominantly inherited erythermalgia was double heterozygous for two separate SCN9A mutations. These data establish primary erythermalgia as a neuropathic disorder and offers hope for treatment of this incapacitating painful disorder.

Investigation of Platelet Functions in Chronic Myeloproliferative Disorders by Optical and Luminesance Method.

Background: Bleeding and thrombosis are common causes of morbidity and mortality in patients with myeloproliferative disorders (MPD). Qualitative platelet abnormalities are frequently found in these patients and range from platelet hypofunction as demonstrated by defective invitro platelet aggregation, acquired storage pool disease and/or platelet membrane defects, in addition to enhanced platelet aggregation, increased plasma beta thromboglobulin levels or shortened platelet survival. In this study we aimed to perform platelet aggregation studies by optical method (on platelet rich plasma=PRP) and luminesance method (on whole blood) in chronic myeloproliferative disorders.Methods:A total of twenty-five patients with MPD (17 chronic myeloid leukemia, 6 polycythemia vera, 2 essential thrombocytosis) were enrolled. Median age was 54,4 (29–76). Platelet aggregation was measured using the optic and luminesance method. The agonists used were adenosine diphosphate (ADP), arachidonic acid (AA) ristocetin and collagen. Platelets were considered to be hyperactive if at least one result (i.e. aggregation or ATP release with one agonist) was above the reference range, and hypoactive if at least one result (i.e. aggregation or ATP release) was below the reference range. Mixed hypo- and hyperactive platelets were considered present when at least one result (i.e. aggregation or ATP release) was below and above the reference range respectively.Results:Platelet aggregation test results by two methods in myeloproliferative disorders were shown in Table 1. The percent for detection of platelet function abnormality by luminesance method was found to be higher than the optic method and a significant difference was shown between two methods (p<0,05).Conclusion Our findings suggest that;1. Luminesance platelet aggregation study is more valuable than optic platelet aggregation study for invitro assessment of platelet function in patients with MPD. 2. The use of luminesance platelet aggregation study appears useful to select patients for antiplatelet therapy.Platelet aggregation test results in myeloproliferative disorders (n=25)NormalHypofunctionHyperfunctionMixTotal abnormalityLuminesance method1(%4)4(%16)8(%32)11(%44)24(%96)Optic method9(%36)11(%44)2(%8)3(%12)16(%64)

Procoagulant surface exposure and apoptosis in rabbit platelets: association with shortened survival and steady-state senescence

The signal(s) for removal of senescent platelets from the circulation are not fully understood; phosphatidylserine (PS) expression on platelets and another marker of apoptosis, loss of mitochondrial inner membrane potential (DeltaPsim), have been implicated in platelet clearance. To investigate whether shortened platelet survival and steady-state platelet senescence are associated with increased surface exposure of PS and DeltaPsim collapse. Survival of in-vitro biotinylated rabbit platelets treated with thrombin or Ca(2+)-ionophore A23187 was tracked by flow cytometry after injection. Steady-state platelet senescence was investigated by infusing biotin to label a platelet cohort. PS expression and DeltaPsim of in-vitro biotinylated platelets and of the aging platelet cohort biotinylated in-vivo were measured by flow cytometry using annexin V-FLUOS and the DeltaPsim-sensitive dye CMXRos, respectively. Although PS expression, DeltaPsim and survival of thrombin-degranulated platelets were similar to those of control platelets, increasing concentrations of A23187 caused increased surface exposure of PS and progressive shortening of platelet survival; only one-sixth of PS-expressing platelets also exhibited DeltaPsim loss. The cohort of senescent, biotinylated platelets remaining in the circulation at 96 h had increased exposure of PS and collapsed DeltaPsim; of the 17% of PS-expressing platelets, one-third did not exhibit DeltaPsim loss. There was also an increase in platelets with collapsed DeltaPsim but not expressing PS. Platelets with shortened survival and senescent platelets have increased surface exposure of PS, that may be involved in their clearance. PS expression can occur independently of DeltaPsim collapse and conversely, in aged platelets, DeltaPsim loss can occur independently of PS expression.

Platelet activation and antiplatelet therapy in patients with ischemic stroke

In patients with ischemic stroke, findings of platelet activation are frequent in platelet function tests. They include increases in plasma levels of beta-thromboglobulin and platelet factor 4, shortening of platelet survival and increasing of platelet lysis, enhancing of shear-induced platelet aggregation (SIPA), and increasing of reticulated platelets as well as platelet fibrinogen binding (PFB) and P-selectin expression (PSE). Aspirin can reduce vascular events including stroke, myocardial infarction and vascular death in high-risk patients with occlusive vascular disease. Aspirin's effect on vascular events is J-shaped, which appeared to be related to the effects on platelet aggregation and release reaction as well as prostaglandin synthesis. Thienopyridines can reduce vascular events even more than aspirin. SIPA was inhibited by thienopyridines but not by aspirin. Combination of aspirin and dipyridamole has an additive effect on preventing subsequent stroke. Aspirin enhanced an inhibitory effect of dipyridamole on SIPA in whole blood. PFB and PSE were not inhibited by aspirin but were partially inhibited by ticlopidine, and markedly inhibited by aspirin plus ticlopidine. Glycoprotein (GP) IIb/IIIa inhibitors can block the final common pathway of platelet aggregation. However, PSE and platelet-derived microparticle formation induced by shear stress were never inhibited at lower concentrations and were rather enhanced at higher concentrations of GP IIb/IIIa inhibitors. These results might be related to the fact that many oral GP IIb/IIIa inhibitors fail to show efficacy in patients with acute coronary syndrome.

Platelet-mediated microvascular inflammation and thrombosis in thrombocythemia vera: a distinct aspirin-responsive arterial thrombophilia, which transforms into a bleeding diathesis at increasing platelet counts

Erythromelalgia is the main, pathognomonic and presenting symptom in patients with essential thrombocythemia and thrombocythemia associated with polycythemia vera. Complete relief from erythromelalgic and acrocyanotic pain is obtained with the cyclooxygenase inhibitors aspirin and indomethacin, but not with sodiumsalicylate, dipyridamol, sulfinpyrozone and ticlopedine. Thus, cyclooxygenase metabolites are necessary for erythromelalgia to develop. Local platelet consumption in erythromelalgic areas became evident by the demonstration of arteriolar fibromuscular intimal proliferation and occlusions by platelet-rich thrombi in skin biopsies, by the findings of shortened platelet survival times, significant higher levels of platelet activation markers β-thromboglobulin, thrombomoduline and increased urinary thromboxane B2 excretion in thrombocythemia patients suffering from erythromelalgia. Aspirin treatment of erythromelalgia in thrombocythemia patients resulted in the disappearance of the erythromelalgic, thrombotic signs and symptoms, correction of the shortened platelet survival times, and a significant reduction of the increased levels of β-TG, PF4, TM and urinary TxB2 excretion to normal. Erythromelalgia is frequently preceded or followed by atypical transient neurologic, ocular or coronary ischemic symptoms, which specifically respond to low-dose aspirin or reduction of platelet counts to normal. The broad spectrum of acropareshesias, erythromelalgia and acrocyanotic ischemia together with the episodic and transient atypical TIAs and ocular or coronary ischemic symptoms are caused by spontaneous activation and aggregation of hypersensitive platelets in the end-arterial microvasculature involving the peripheral, cerebral and coronary circulation of thrombocythemia patients. These microvacular circulation ischemic disturbances in thrombocythemia vera already occur at platelet counts in excess of 400 × 10 9 l –1. Low-dose aspirin is highly effective and safe in the cure and prevention of thrombotic and ischemic events and does not elicit bleedings at platelet counts below 1000 × 10 9 l –1. Spontaneous hemorrhages usually occur at very high platelet counts far in excess of 1000 × 10 9 l –1 (HT) due to an acquired von Willebrand factor deficiency at increasing platelet counts. At platelet counts between 1000 and 2000 × 10 9 l –1, thrombosis and bleeding (ETT and HT) frequently occur in sequence or paradoxically and low-dose aspirin does prevent thrombotic complications but aggravates or may elicit bleeding symptoms. Reduction of the platelet count to below 1000 × 10 9 l –1 by platelet lowering agents usually results in the disappearance of the bleeding tendency and improvement of the von Willebrand syndrome, but the thrombotic tendency persists as long as platelet counts are above the upper limit of normal.

Treatment with megestrol acetate improves human immunodeficiency virus-associated immune thrombocytopenia.

Splenic macrophage Fc gamma receptors participate in the pathophysiology of immune cytopenias, and in such disorders, the beneficial effects of glucocorticoids are in part mediated by decreased expression of macrophage Fc gamma receptors. In the animal model, progesterones, like glucocorticoids, inhibit expression of these receptors. Megestrol acetate (MA) is a progesterone frequently used for treating human immunodeficiency virus (HIV)-associated anorexia-cachexia. Twenty-eight patients with HIV-associated thrombocytopenia with shortened platelet survival and increased platelet-associated immunoglobulin G (IgG) who were being treated with MA for anorexia-cachexia were prospectively studied for a 6-month period to assess the potential role of progesterones in the treatment of immune thrombocytopenia. Treatment with MA for non-consecutive periods of 2 months and 1 month significantly increased platelet count and platelet survival without significant alteration of platelet-associated immunoglobulin levels. Of the 28 patients studied, 22 presented a complete response, 19 presented a complete response 1 month after finishing the MA treatment regimen, and 12 remained in complete response for a further month. Expression of Fc gamma receptors (Fc gamma RI and Fc gamma RII) by peripheral blood monocytes and the in vitro recognition of IgG-sensitized cells by monocytes were significantly decreased by the MA treatment. Decreased expression and functioning of these receptors significantly correlated with platelet counts and survival times, but no relationship was found with platelet-associated immunoglobulin, circulating immune complexes, body mass index, plasma HIV load, or CD4 lymphocyte levels. These results suggest that treatment with progesterones, like MA, may be an alternative therapy for immune cytopenias, with few side effects.

Novel treatment modalities: new platelet preparations and substitutes.

Keywords: platelets; platelet substitutes; platelet haemostatic function; blood substitutes; platelet function

The Effect of Chloroquine on Circulating Platelet Survival in the Rat.

BACKGROUND: Chloroquine inhibits platelet aggregation. Because platelet aggregation may lead to the lysis of platelets, the effect of chloroquine administration on circulating platelet survival was studied. METHODS AND RESULTS: Platelets harvested from the blood of male inbred (WAG) rats were labeled with (111)Indium oxine and returned to other inbred rats. At timed intervals, blood samples were drawn from the rats and taken for radioactivity estimation. In some experiments, the rats (n = 10) received chloroquine (10 mg/kg) intraperitoneally daily for 3 days. Control rats (n = 10) received chloroquine (10 mg/kg) intraperitoneally daily for 3 days. Control rats (n = 10) received normal saline. Indium-labeled platelets disappeared exponentially in control and test rats. The fraction of indium disappearing/h was significantly less in chloroquine-treated rats than in control rats (0.0368 +/- 0.0016 vs 0.0520 +/- 0.0016, mean +/- SEM; P <.001). In all, 99% of the labeled platelets disappeared in 5.3 days in chloroquine-treated rats and 3.7 days in control rats. CONCLUSIONS: Chloroquine administration increases the life span of circulating platelets in rats. If the results are confirmed in humans, chloroquine may prevent the shortened platelet survival and thrombocytopenia common in malarial infection and other thrombotic disorders.