One of the mechanisms by which platelet-derived microparticles elicit procoagulant activity is by an increased exposure of phosphatidylserine on their surface. We have previously demonstrated the utility of an activated factor X-based assay for the detection of procoagulant phospholipid activity [Xa clotting time (XACT)]. The objective of this study was to further characterize the specificity of the XACT to detect microparticle-associated procoagulant phospholipid activity. XACT testing for procoagulant phospholipid was measured using an ST4 machine and microparticle counting was performed using flow cytometry for Annexin V binding. Plasma microparticle counts were significantly correlated to XACT times (P = 0.0001). The XACT assay was insensitive to tissue factor, whereas the addition of microparticles to a whole blood sample shortened XACT times. Procoagulant phospholipid activity could be detected in both citrate and EDTA anticoagulated samples; however, XACT times and microparticle counts were more stable in EDTA anticoagulated samples over a 60 min period. The procoagulant phospholipid activity of microparticles generated by collagen stimulation was significantly impaired in EDTA anticoagulated samples when compared with citrate. Microparticles were capable of higher degrees of thrombin generation than equivalent concentrations of phosphatidylserine (as assessed by XACT times), suggesting that other factors bound to the microparticle surface enhance the procoagulant response. In conclusion, the XACT assay is a specific method for the detection of procoagulant phospholipid activity arising from phosphatidylserine on the microparticle surface; however, other factors presumably bound to the surface of the microparticle may also contribute to enhanced thrombin generation detectable by prothrombinase assays.
Cyclic thrombocytopenia (CT) is a rare condition characterized by periodic fluctuations in the platelet count, resulting in periods of thrombocytopenia and in some cases, rebound thrombocytosis. It is often misdiagnosed as immune thrombocytopenic purpura (ITP), and many patients will have received and failed a number of standard ITP treatments before a diagnosis of CT is suspected and made. Although some patients with CT require close observation only, other patients require therapeutic intervention and this may include medications such as danazol, cyclosporine and in more recent years, thrombopoietin (TPO)-mimetic agents. We describe a 32-year-old female with CT and associated marked rebound thrombocytosis who was originally considered to have ITP. Endogenous levels of TPO and numbers of reticulated platelets also underwent periodic fluctuations, and for a prolonged period of time, marked thrombocytopenia without cycling was observed. After a period of 4 months, platelet cycling spontaneously resumed. There was an association between platelet cycling and the hormonal cycle, and in addition, neutrophil and lymphocyte counts were also observed to undergo periodic fluctuations. A 32-year-old female was originally referred to our institution for an opinion regarding longstanding thrombocytopenia presumed to be due to ITP, it was first diagnosed when the patient was 19-years-old. A bone marrow biopsy performed at initial diagnosis demonstrated increased numbers of megakaryocytes including immature forms. The patient subsequently received several therapies for ITP including steroids, intravenous immunoglobulin and danazol, and had also undergone a splenectomy. The platelet response was limited and unsustained over the succeeding years, and the patient described an apparent cyclical nature to her thrombocytopenia with intervening periods of extreme thrombocytosis. Bleeding symptoms included episodes of significant bruising, epistaxis, and oral blood blisters. Oral tranexamic acid was used by the patient during bleeding episodes and they were relatively mild and short-lived and required no further intervention. The patient had irregular menses with periods of prolonged amenorrhea. Any episodes of iron deficiency were managed with oral iron supplementation. There was no excessive bleeding with delivery of her two children and there were no other pregnancies or history of miscarriages. There were no documented thrombotic events or sypmtoms to suggest ischemia during the periods of rebound thrombocytosis. Other history included beta thalassemia trait, asthma, and polycystic ovarian syndrome. There was no clinical evidence of an underlying auto-immune disorder although antinuclear antibodies were intermittently positive on several occasions. JAK2 V617F gene mutational testing was negative and platelet-specific antibodies were not detected. We initially investigated the patient over two complete cycles, and then continued monitoring full blood counts for approximately 2 years. Over the first two cycles, platelet count, reticulated platelet counts, serum TPO, and hormone levels were assessed. The patient was not on any therapy designed to increase the platelet count during this entire period, however did receive clomiphene for some time. The platelet count varied from 8 × 109/L to 1,665 × 109/L throughout a single cycle (Fig. 1). The cycle length was approximately 30 days, during that time the platelet count was less than 20 × 109/L for a total of around 10 days. During the 2 year assessment, there was a period of 120 days where there was no recorded increase in the platelet count above 25 × 109/L. A bone marrow biopsy performed during this period (day 178) demonstrated increased numbers of megakaryocytes that were of normal morphology and no karyotypic abnormality was found. A spontaneous resumption to the platelet cycling occurred after day 240 and it was considered that the persistent thrombocytopenia may have been due to concomitant clomiphene therapy that the patient began taking during this period. Fluctuations in platelet count. (a) The patient's platelet count is plotted over a 680 day period. The grey shaded area represents the normal range (150–400 × 109 platelets/L). Over a 60-day period (which equated to two consecutive cycles), it was also noted that there was a fluctuation in both the lymphocyte and neutrophil count, and the peak in leukocytes appeared to precede the increase in platelet count (Fig. 2). In addition, there were other peaks in the lymphocyte count which were not accompanied by an increase in either the neutrophil or platelet counts. The relationship between platelet, lymphocyte and neutrophil counts and thrombopoeitin levels over a 50-day period. (a) The patient's platelet count (closed circles) is plotted with the neutrophil count (open circles) and lymphocyte count (open squares). (b) The patient's platelet count (closed circles) is plotted along with the patient's thrombopoeitin (TPO) levels (open circles). Reticulated platelet counts are shown as the shaded region. Serum TPO levels (normal range: 0–228 pg/mL) and numbers of reticulated platelets also demonstrated a cyclical variation over the 60-day period (Fig. 2). TPO levels were inversely related to the platelet count and interestingly, the serum TPO concentration decreased several days before the increase in platelet count. Absolute numbers of reticulated platelets increased during rises in the platelet count and in keeping with TPO levels, the reticulated platelet count appeared to decrease before the decrease in the total platelet count. The relationship between serum hormone levels and platelet and neutrophil counts was assessed over the 60-day period of two cycles (Fig. 3). Cyclical variations in human luteinizing hormone (HLH) levels were almost synchronous with the variations in platelet counts, whilst estradiol levels were inversely related to platelet cycling. The relationship between platelet and neutrophil counts and hormone levels over a 120 day period. The patient's platelet count (closed circles), neutrophil count (open circles), and hormone levels (follicle stimulating hormone—FSH, human luteinizing hormone—HLH, progesterone, and estradiol) were assessed over a 120-day period (four cycles). CT is a relatively uncommon disorder and, a recent review highlights the clinical features and therapeutic strategies in 51-reported cases [1]. Most cases of CT appear to be idiopathic, whilst some are secondary to myelodysplastic and myeloproliferative disorders. CT is more frequent in females and in the majority of premenopausal women there is a relationship between the platelet cycle and the menstrual phase. Whilst the pathophysiology of CT is unclear, a hormonal etiology is considered possible. A number of therapeutic interventions have been used without effect; however there is anecdotal success with hormonal therapy including danazol and progestins. Recently, a number of cases have been successfully treated using TPO-mimetic agents [2]. This patient presented with features initially considered to be ITP, however after a period of many years and close observation, was found to have CT. In addition to the thrombocytopenia, the patients' cycle was characterized by marked rebound thrombocytosis, a feature which is considered unique to CT. The prolonged period of marked thrombocytopenia appeared to coincide with the use of clomiphene which the patient took for a number of months. Clomiphene is a selective estrogen receptor modulator, most commonly used to enhance fertility. Its primary site of action is the hypothalamus, where it blocks the negative feedback effect of circulating endogenous estrogen and results in elevated levels of follicle stimulating hormone (FSH) and HLH. Thus, it is not surprising that the administration of clomiphene in this patient affected her platelet cycling. During the period of prolonged thrombocytopenia, a bone marrow biopsy revealed plentiful to increased megakaryocytes that were of normal morphology. Previous reports have described abundant bone marrow megakaryocytes at the platelet peak [3], but few at the platelet nadir [3, 4] in patients with CT. Zent et al. described normal megakaryocyte precursors before the platelet peak, but found that serum from a CT patient inhibited the growth of megakaryocyte precursors from both the patient and from a normal donor. A cyclical nature was also found in neutrophil and lymphocyte counts, with the peak neutrophil count occurring days before the peak platelet count, whilst multiple lymphocyte peaks were seen. Whilst also a rare disorder, cyclic neutropenia was been well documented [5, 6], an autosomal dominant disorder most typically arising from a mutation in neutrophil elastase (ELA2) [7, 8]. As described in previous case reports [3, 9-12], there was an inverse relation between platelet counts and TPO levels in this patient. We did however note that TPO concentrations began to decrease several days before the increase in platelet counts. This may suggest an increase in the numbers of megakaryocytes in the days preceding an increase in platelet count. Furthermore, during the period in which there was no recorded increase in platelet count, the bone marrow contained elevated number of megakaryocytes, whilst the TPO concentration was 176 pg/mL. As the patients TPO levels extended above the normal range during periods of thrombocytopenia, it was felt that the administration of TPO-mimetic agents would not provide any benefit to the patient. Additionally, as the patient typically presented with a rebound thrombocytosis, it was felt that TPO-mimetic agents might further extend the thrombocytosis. As well as fluctuations in TPO levels, previous reports have described synchronous fluctuations in IL-6 [13], IL-7 [3], SCF [3], and GM-CSF [13] TGF-B1 [3], glycocalin [10], with reciprocal fluctuations of M-CSF [3]. Near the end of the first platelet peak, the absolute number of reticulated platelets began to decrease before the decrease in the total platelet count. This may suggest a failure in the production of platelets and/or megakaryocytes, or a failure in the release of platelets from the bone marrow. This patient displayed a relationship between platelet cycling and the hormonal cycle. A possible relationship between female hormonal levels and platelet cycling has been noted by others [1, 4]. The female preponderance of this disorder and the relatively good response to hormonal therapy with progestin-like effects support this [1]. In addition, it has been reported that estrogenic hormones can modulate expression of monocyte Fc gamma receptors in patients with CT and platelet auto-antibodies, thus altering platelet survival. Although many cases of CT have been reported to occur in line with the menstrual cycle, CT may also exist in males [14, 15] and in postmenopausal females. In this case report, we have identified a case of CT in a 32-year-old female experiencing periods of thrombocytopenia and periods of thrombocytosis. The changes in the platelet count appeared to be inversely related to serum TPO levels, but it was apparent that the TPO levels began to decrease several days before the rise in platelet count. During our investigation, she experienced a 4-month period in which there was no detected rise in the platelet count. The patient did not receive any therapy intended to increase the platelet count, largely because of the spontaneous rebound thrombocytosis and the concern of potential adverse effect of an even higher platelet count. Serum samples were collected using serum separator tubes. TPO levels were measured using a commercially available ELISA (Quantikine, RND Systems) that measures the free TPO concentration in serum samples. The percentage of reticulated platelet counts was determined in EDTA-anticoagulated whole blood by flow cytometry using thiazole orange staining. This percentage was then applied to the platelet count to obtain an absolute reticulated platelet count. Five microliter of EDTA anticoagulated whole blood was added to 40-μL phosphate buffered saline solution (PBS) and 5-μL anti-CD41a-PerCP-Cy5.5 and incubated for 20 min before the addition of 1-mL PBS containing thiazole orange. This was incubated for a further 30min, before centrifugation at 1,500g for 5 min. The top 900 μL was removed and replaced with 900 μL PBS, before analysis by flow cytometry. Samples were analyzed with respect to 0.5% of events in tube processed without thiazole orange. David Ewan Connor* , Joanne Emily Joseph* , * Department of Haematology and Haematological Stem Cell Transplantation, St Vincent's Hospital, Sydney, Australia;, St Vincent's Clinical School, Faculty of Medicine, The University of New South Wales, Sydney, Australia.
Summary Previous studies report conflicting results concerning the potential significance of thrombophilic genotypes in postarthroplasty venous thromboembolism (VTE). This study assessed thrombophilic genotypes, haemostatic and clinical variables as independent risk factors for VTE postarthroplasty. A total number of 569 patients undergoing elective lower limb arthroplasty at a single centre were prospectively studied. All patients were interviewed and had blood samples collected preoperatively. Bilateral lower limb ultrasonography was performed at day 7 ± 2 postoperatively in all patients (ventilation/perfusion lung scanning in symptomatic patients only). The incidence of inhospital postoperative VTE was 26%. In univariate analysis – increased age, knee arthroplasty, recent surgery, general anaesthesia, shorter operation time, non‐receipt of blood transfusion and differences in surgical practice (including use of pneumatic calf compression, surgical drains and postoperative bandaging techniques) were significantly associated with VTE. Factor V Leiden, prothrombin G20210A and MTHFR C677T mutations were not significant risk factors for VTE, and of all haemostatic variables tested, only median activated partial thromboplastin time showed significant difference between VTE and non‐VTE patients (34 s vs. 33 s). Multiple logistic regression analysis demonstrated that increased age, knee arthroplasty and individual surgeon's routine practices were the only significant independent risks for VTE; hence routine preoperative blood screening for a potential hypercoaguable state is not indicated in this surgical setting.
Atherosclerosis remains a leading cause of morbidity and mortality, with revascularization remaining a cornerstone of management. Conventional revascularization modalities remain challenged by target vessel reocclusion-an event driven by mechanical, thrombotic, and proliferative processes. Despite considerable advancements, restenosis remains the focus of ongoing research. Adjunctive agents, including dipyridamole, offer a multitude of effects that may improve vascular homeostasis. We sought to quantify the potential therapeutic impact of dipyridamole on vascular occlusion. We performed a literature search (EMBASE and MEDLINE) examining studies that encompassed 3 areas: (1) one of the designated medical therapies applied in (2) the setting of a vascular intervention with (3) an outcome including vascular occlusion rates and/or quantification of neointimal proliferation/restenosis. The primary outcome was vascular occlusion rates. The secondary outcome was the degree of restenosis by neointimal quantification. Both human and animal studies were included in this translational analysis. There were 6,839 articles screened, from which 73 studies were included, encompassing 16,146 vessels followed up for a mean of 327.3 days (range 7-3650 days). Preclinical studies demonstrate that dipyridamole results in reduced vascular occlusion rates {24.9% vs. 48.8%, risk ratio 0.53 [95% confidence interval (CI) 0.40-0.70], I2 = 39%, P < 0.00001}, owing to diminished neointimal proliferation [standardized mean differences -1.13 (95% CI -1.74 to -0.53), I2 = 91%, P = 0.0002]. Clinical studies similarly demonstrated reduced occlusion rates with dipyridamole therapy [23.5% vs. 31.0%, risk ratio 0.77 (95% CI 0.67-0.88), I2 = 84%, P < 0.0001]. Dipyridamole may improve post-intervention vascular patency and mitigate restenosis. Dedicated studies are warranted to delineate its role as an adjunctive agent after revascularization.
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