Rapid determination of anti-heparin/platelet factor 4 antibody titers in the diagnosis of heparin-induced thrombocytopenia

PURPOSE: Heparin-induced thrombocytopenia is mediated by antibodies directed against the heparin-platelet factor 4 (heparin/PF4) complex. Our aim was to investigate whether rapid measurement of anti-heparin/PF4 antibodies could improve the diagnostic workup of patients with suspected heparin-induced thrombocytopenia. METHODS: We examined 148 consecutive patients in our laboratory between January 1995 and June 2001 for suspected heparin-induced thrombocytopenia. Clinical data allowed retrospective assessment of the likelihood of heparin-induced thrombocytopenia. Antibodies against the heparin/PF4 complex were detected by a rapid particle gel immunoassay. RESULTS: Anti-heparin/PF4 antibodies were detected in 69 (47%) of the 148 patients, at dilution titers from 1 to 256. Clinically "likely" or "very likely" heparin-induced thrombocytopenia was significantly more common in patients with titers >or=4 (95% [39/41]) than in those with undetectable antibodies (13% [9/70]; P <0.0001), a titer of 1 (18% [4/22]; P <0.0001), or a titer of 2 (33% [2/6]; P = 0.001). All 19 samples with a positive platelet aggregation test had anti-heparin/PF4 antibody titers of at least 4, including 15 samples with titers >or=32. Thromboembolic complications in heparin-treated patients were significantly more prevalent in patients with titers >or=4 (63% [26/41]) than in those with undetectable antibodies (8% [6/79]; P <0.0001) or a titer of 1 (9% [2/22]; P <0.0001). Of the 11 patients with a titer of 1 who were maintained on heparin, none developed worse thrombocytopenia or thromboembolic complications. CONCLUSION: Anti-heparin/PF4 antibody titers, which can be measured rapidly and reproducibly using a particle gel immunoassay, can be used as a confirmatory test to complement a clinical likelihood score among patients with suspected heparin-induced thrombocytopenia.     

Heparin-releasable platelet factor 4 in patients with coronary artery disease.

Recently, platelet factor 4 (PF4) release by heparin (heparin-releasable PF4) has been examined as a useful marker of the interaction between the substances liberated from circulating platelets and the vascular endothelium. We compared the plasma levels of PF4 and beta-thromboglobulin (beta-TG) after intravenous heparin injection in patients with coronary artery disease (CAD) and normal control subjects. We also studied the effects of low-dose aspirin (81 mg/day) on the plasma level of heparin-releasable PF4 in the CAD patients. Blood samples were obtained before and 5 min after the intravenous injection of heparin (1,000 IU) from 23 patients with CAD and 15 normal control subjects. Although the plasma beta-TG level remained unchanged after heparin injection, the plasma PF4 level markedly increased in both groups. There was a significant difference in plasma PF4 levels at 5 min after heparin injection between the CAD group (100.1 +/- 38.1) and the control group (61.0 +/- 24.0) (p less than 0.01). The PF4/beta-TG ratio after heparin injection was also higher in the CAD group than in the control group (p less than 0.01). There was a correlation between the PF4/beta-TG ratio after heparin and the Gensini CAD score, which defines the severity of coronary atherosclerosis (r = 0.489, n = 23, p less than 0.01). Low-dose aspirin was administered to 11 CAD patients for 246.0 +/- 28.8 days. Blood samples for the assay of PF4 and beta-TG were obtained as stated above, and platelet aggregation, thromboxane B2 (TxB2), and 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) levels were also measured before and during aspirin administration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Affinity purification of heparin-dependent antibodies to platelet factor 4 developed in heparin-induced thrombocytopenia: biological characteristics and effects on platelet activation

Antibodies to heparin platelet factor 4 (H-PF4) complexes were purified from the plasma of three patients with heparin-induced thrombocytopenia (HIT) using affinity chromatography. From each plasma, the largest amount of antibodies was eluted with 2 M NaCl at pH 7.5 (peak 1) and the remainder was obtained using 0.1 M glycine/0. 5 M NaCl at pH 2.5 (peak 2). In an enzyme-linked immunosorbent assay (ELISA), we then showed that each patient had developed antibodies to PF4 displaying different characteristics. In patient 1, peak 1 IgG reacted almost exclusively with H-PF4 complexes, whereas peak 2 IgG had similar reactivity with PF4 whether or not heparin was present. Patient 2 expressed a mixture of IgA, IgM and IgG and both fractions bound to PF4 alone or to H-PF4 complexes. Finally, IgG in patient 3 only bound to H-PF4 and was unreactive with PF4 alone. Using [14C]-serotonin release assays, the antibodies developed in the three patients and exhibiting the strongest ability to activate platelets with heparin were those having the highest affinity to H-PF4. These results strongly support the hypothesis that HIT antibodies to PF4 are heterogeneous regarding their affinity and specificity for target antigens and this may greatly influence their ability to activate platelets and their pathogenicity.     

2型糖尿病患者大血管病变与活化血小板的相关性研究

目的探讨2型糖尿病患者活化血小板与大血管病变的关系。方法将江苏省苏北人民医院2002-05~2004-11收治的98例2型糖尿病患者,按有无大血管病变分为大血管病变组50例、单纯糖尿病组48例;另设50名正常人为对照组。采用流式细胞仪检测活化血小板标记物血小板膜糖蛋白(PAC-1、CD62P)的表达以及糖化血红蛋白(HbA1c)、C反应蛋白(CRP)、空腹血糖(FBG)、空腹C肽值(C-P)、甘油三酯(TG)、胆固醇(TC)、低密度脂蛋白(LDL-C)、高密度脂蛋白(HDL-C)等,做相关性分析。结果单纯2型糖尿病患者活化血小板标志物的阳性表达率比正常对照组明显升高(P<0·05),大血管病变组活化血小板标志物的阳性表达率比单纯2型糖尿病组升高更明显(P<0·01)。相关分析中,活化血小板的阳性表达率与LDL-C呈明显正相关。结论活化血小板标志物的阳性表达率与2型糖尿病大血管病变发生呈明显相关,LDL-C水平的增高是导致动脉粥样硬化的独立危险因素。     

Platelet factor 4 release during exercise in patients with coronary artery disease

Many recent studies provide evidence that increased platelet activation occurs in a significant number of patients with atherosclerotic coronary artery disease. The mechanisms responsible for this activation are unknown, although there have been studies suggesting a correlation with abnormal lipoproteinemia, acute myocardial infarction, unstable angina, and exercise-induced myocardial ischemia. We studied 84 patients undergoing standardized treadmill exercise using either a Bruce [N = 63] or symptom-limited Naughton protocol [N = 21]. In contrast to ten healthy volunteer subjects, the patient group demonstrated a significant increase in plasma concentrations of platelet factor 4 [PF4] between pre- and postexercise blood samples confirming earlier reports of exercise-induced platelet activation and secretion. As with previous studies, however, only a subset of patients demonstrated this response. When the entire group was analyzed for the presence or absence of electrocardiographic ischemic changes and the presence of documented versus suspected coronary artery occlusions, there were no differences noted between groups that explained the variable responses measured. However, there was a significant difference between patient groups when analyzed by whether or not they were being treated with β-blocking agents. Patients who were being treated with propranolol or one of the longer-acting β-blocking agents did not have a significant increase in plasma PF4 following exercise, in contrast to patients who were not β-blocked. Plasma concentrations of epinephrine, norepinephrine, and lactic acid were measured in 49 patients and all normal subjects. There was no correlation between the changes in plasma PF4 concentrations and any of these three variables, suggesting that platelet activation was not occurring through direct platelet activation by circulating catecholamines. This study provides further evidence that there is a subset of CAD patients with platelet hyperactivity. This is the first time that β-blockade has been demonstrated to modify this platelet response. The effectiveness of β-blocking agents in CAD may be in part related to their antiplatelet effect.     

哮喘大鼠支气管肺泡灌洗液中血管内皮生长因子和血小板源性生长因子的变化及缬沙坦的干预

目的观察血管内皮生长因子（VEGF）和血小板源性生长因子（PDGF）在哮喘大鼠支气管肺泡灌洗液（BALF）中的变化,探讨两者与哮喘气道重塑的关系以及缬沙坦的干预作用。方法50只Wistar大鼠随机分成5组,每组10只：A组（正常对照组）、B组（哮喘组）、C组[15mg／（kg·d）缬沙坦]、D组[30mg／（kg·d）缬沙坦]和E组[50mg／（kg·d）缬沙坦]。分别观察各组肺组织切片的病理改变,用酶联免疫吸附法（EHSA）检测各组大鼠BALF中VEGF和PDGF的水平。结果哮喘大鼠肺组织出现了气道重塑的病理改变;与A组比,B组BALF中VEGF和PDGF水平显著增高（P〈0．01）,与B组比,缬沙坦干预后的C组、D组和E组BALF中VEGF和PDGF均显著减少（P〈0．05或P〈0．01）。结论VEGF和PDGF在哮喘大鼠BALF中浓度增高,可能参与了哮喘发病和气道重塑过程。缬沙坦能明显降低哮喘大鼠BALF中VEGF和PDGF的水平,有助于改善哮喘大鼠气道重塑的病理生理过程,其作用机理还有待进一步研究。     

Human parvovirus PARV4 in clotting factor VIII concentrates

BACKGROUND AND OBJECTIVES: Parvoviruses are small non-enveloped DNA viruses, relatively resistant to virus inactivation procedures. The recently identified human parvovirus PARV4, including a related genotype 2 virus (also termed PARV5), has been found to be a contaminant of pooled plasma used in the manufacture of plasma-derived products. This report describes an investigation to determine whether PARV4 is present in clotting factor concentrates. MATERIALS AND METHODS: Factor VIII concentrates manufactured in the past 30-35 years were screened for PARV4 and human parvovirus B19 (B19V) sequences. Viral loads in products testing positive for PARV4 were quantified using a consensus TaqMan assay designed to a highly conserved region. DNA sequence analysis was performed to confirm the genotypes present. RESULTS: From a total of 175 lots of factor VIII concentrate, 28 of these contained PARV4 sequences, and in two lots both genotypes 1 and 2 were found to be present. The highest viral loads observed exceeded 10(5) copies per ml. The majority of factor VIII concentrates testing positive for PARV4 were manufactured in the 1970s and 1980s. Human B19V was also a frequent contaminant of these products. CONCLUSIONS: PARV4 was detected in 16% of factor VIII concentrates, particularly in older batches from the 1970s and 1980s. The significance in terms of the viral safety and potential transmission to recipients of these products is not yet known.     

Development of a high-yield expression and purification system for platelet factor 4

Heparin-induced thrombocytopenia (HIT) is an adverse drug reaction characterized by IgG antibodies bound to complexes of platelet factor 4 (PF4) and heparin. The majority of diagnostic tests for HIT rely on an exogenous source of PF4 to identify anti-PF4/heparin antibodies. These include the PF4-dependent enhanced serotonin release assay (PF4-SRA) among others. Using a bacterial expression system, we developed a novel and efficient method of producing recombinant human PF4 (rhPF4) that is biochemically and antigenically similar to platelet-derived human PF4. rhPF4 was produced using the pET expression system in the BL21(DE3) strain of Escherichia coli. The system was optimized for protein expression using isopropyl β-D-1-thiogalactopyranoside at different induction temperatures and incubation times. rhPF4 solubility was improved by using different detergents during cell lysis and by purifying with heparin affinity and ion exchange chromatography. Biochemical characteristics of rhPF4 were investigated using mass spectrometry, SDS-PAGE analysis, and gel filtration chromatography and compared to platelet-derived PF4. Antigenic and functional characteristics of rhPF4 were studied using the anti-PF4/heparin EIA and the PF4-SRA. Using this method, we could produce 11.4 ± 0.6 mg of pure rhPF4 per liter of bacterial culture. Absorbance readings from the anti-PF4/heparin EIA using platelet-derived and rhPF4 were highly correlated (n = 194; r = 0.9545, p < 0.0001); and functional release of serotonin in the PF4-SRA induced by anti-PF4/heparin antibodies was similar to either platelet-derived or rhPF4 and heparin (r = 0.9597, p < 0.0001). Our method of rhPF4 production is efficient and does not rely on a source of platelets. The rhPF4 purification method described produces greater yields at a lower cost than other current methods. The application of this method can improve the efficiency of biochemical investigations and HIT diagnostic testing by supplying sufficient amounts of PF4.     

缺血性脑血管病患者高血压时程及相关影响因素分析

目的研究有高血压史的缺血性脑血管病(ischemiccerebralvasculardisease,ICVD)人群中,高血压发生与ICVD发生的时程关系及影响因素。方法回顾性研究1990～1999年间解放军总医院神经内科收治的1166例首次ICVD发生之前或当时发现高血压的病人,确定高血压时程,即发现高血压至首次ICVD发生时间间隔,采用多元线性回归模型分析影响高血压时程长短的因素。结果高血压时程范围0～60(14.29±10.63)年,中位数11年。多元线性回归分析显示发现高血压年龄增长,吸烟、糖尿病使高血压时程缩短;军人的高血压时程比农民长;治疗高血压较不治疗者时程延长。结论发现高血压至首次ICVD发生的时间间隔变动较大,半数患者在发现高血压11年内发生首次ICVD,高血压与吸烟和糖尿病在导致ICVD发生的过程中具有累积效应,降压治疗能够推迟ICVD的发生。     

PEGylation prevents bacteria-induced platelet activation and biofilm formation in platelet concentrates

Bacterial contamination of platelet concentrates represents the greatest post-transfusion infectious risk. Biofilm formation in this environment resulting from platelet-bacteria interactions can lead to non-uniform contaminant distribution and thus missed detection. As formation of platelet-bacteria aggregates is largely based on receptor-ligand interactions, we examined whether shielding these events would result in reduced biofilm formation by contaminant bacteria. We introduced methoxypoly(ethylene glycol) to covalently modify the platelet surface using a process termed 'PEGylation'. In the first study of its kind, we demonstrate that PEGylated platelet concentrates inoculated with Staphylococcus epidermidis display a significant reduction in bacterial binding and biofilm formation.     

血管内皮生长因子在脑缺血中的作用

血管内皮生长因子(vascular endothelial growth factor,VEGF)是一种重要的调节多种内皮功能的血管生长因子.脑缺血后,VEGF不仪能促进血管内皮细胞增殖和迁移,参与血管生成,增加血管通透性,而且在神经保护和神经发生等方面也起着重要作用.文章就VEGF在缺血性脑损伤中的作用进行了综述.     

Effect of heparin on in vivo platelet factor 4 (PF4) release and platelet aggregation after aspirin administration

We studied the release in vivo of platelet specific proteins platelet factor 4 (PF4) and β-thromboglobulin (βTG), and confirmed that only PF4 is released, after heparin intravenous administration. A good correlation was found between platelet count and PF4 and this seems at variance with the idea that the source of released PF4 is vascular endothelium rather than platelets; however, such a possibility cannot be excluded. Although platelet function was blocked by aspirin injection heparin was still able to induce the release of PF4 and exerted a potentiating effect on platelet aggregation induced by ADP and collagen.     

Lipoprotein (a) and vascular disease in diabetic patients

Summary In order to assess the potential role of lipoprotein(a) as a risk factor for cardiovascular disease in diabetes mellitus, plasma concentrations were measured in a large group (n=500) of non-insulin-dependent (NIDDM, n=355) and insulin-dependent (IDDM, n=145) patients. Concentrations of lipoprotein (a) were compared in diabetic patients with (n=153) or without (347) documented vascular disease (ischaemic heart disease, peripheral vascular disease or macroangiopathy). They were significantly higher (p<0.05) in patients with ischaemic heart disease (mean [interquartile range] 15.5 (5.0–38.0) vs 9.0 (4.5–26.0) mg/dl) or macroangiopathy (13.0 (5.0–38.0) vs 9.0 (4.0–25.0) mg/dl) compared to patients without manifestations of vascular disease. In addition, stepwise logistic regression analysis identified lipoprotein (a) levels 30 mg/dl as being independently associated with the presence of cardiovascular disease. Lipoprotein (a) was an independent risk factor for ischaemic heart disease and macroangiopathy in this group of IDDM and NIDDM patients.Abbreviations Lp(a) lipoprotein (a) - LDL low density lipoproteins - CVD cardiovascular disease - IHD ischaemic heart disease - PVD peripheral vascular disease - HDL high density lipoproteins     

睡眠呼吸暂停综合征患者白天嗜睡与脑血管疾病发生的关系

目的探讨睡眠呼吸暂停综合征（SAS）患者白天嗜睡是否与脑血管疾病的发生有关。方法对36例SAS合并短暂性脑缺血发作（合并TIA组），40例SAS合并高血压（合并高血压组）和42例无合并症的SAS（单纯SAS组）患者进行多导睡眠监测，采用Epworth嗜睡评估表（ESS）对其白天嗜睡程度进行测评，同时行全血黏度和红细胞压积测定。结果与单纯SAS组相比，合并TIA组和合并高血压组体质量指数（BMI）、颈围、腰臀比（WHR）、ESS、呼吸暂停低通气指数（AHI）、微觉醒指数（MAI）明显升高（P均〈0．05），合并TIA组与合并高血压组之间ESS、MAI差异无显著性；AHI、最长呼吸暂停时间，合并TIA组明显大于合并高血压组及单纯SAS组（P均〈0．05），全血黏度和红细胞压积，合并TIA组、合并高血压组明显高于单纯SAS组（P均〈0．05）。结论SAS患者白天嗜睡对脑血管疾病发生有预测意义。     

The complicated relationships of heparin-induced thrombocytopenia and platelet factor 4 antibodies with COVID-19

COVID-19 (coronavirus disease 2019) represents a prothrombotic disorder, and there have been several reports of platelet factor 4/heparin antibodies being present in COVID-19-infected patients. This has thus been identified in some publications as representing a high incidence of heparin-induced thrombocytopenia (HIT), whereas in others, findings have been tempered by general lack of functional reactivity using confirmation assays of serotonin release assay (SRA) or heparin-induced platelet aggregation (HIPA). Moreover, in at least two publications, data are provided suggesting that antibodies can arise in heparin naïve patients or that platelet activation may not be heparin-dependent. From this literature, we would conclude that platelet factor 4/heparin antibodies can be observed in COVID-19-infected patients, and they may occur at higher incidence than in historical non-COVID-19-infected cohorts. However, the situation is complex, since not all platelet factor 4/heparin antibodies may lead to platelet activation, and not all identified antibodies are heparin-dependent, such that they do not necessarily reflect “true” HIT. Most recently, a “HIT-like” syndrome has reported in patients who have been vaccinated against COVID-19. Accordingly, much more is yet to be learnt about the insidious disease that COVID-19 represents, including autoimmune outcomes in affected patients.     

Association of interarm blood pressure difference with cardio‐cerebral vascular disease: A community‐based,cross‐sectional study

Interarm blood pressure difference (IAD) is a risk factor for peripheral artery disease and cardio‐cerebral vascular disease (CCVD). The current study examines the association of IAD with stroke and coronary heart disease in a Chinese community. A cross‐sectional study was conducted in Pudong New Area in Shanghai, China. A total of 10 657 residents aged 15 years and older were randomly selected through three‐stage sampling. Volunteers had systolic and diastolic blood pressure (BP) measured in both arms at recruitment, and IAD was defined in both arms as the absolute difference in BP. Medical records of study participants were reviewed by investigators to confirm measurements. Logistic regression models were used to assess the association between systolic interarm blood pressure difference (sIAD) and diastolic interarm blood pressure difference (dIAD) with stroke and coronary heart disease. Compared with dIAD <5 mm Hg, the multivariate adjusted odds ratio (OR) of stroke prevalence was 1.357 (95% CI 0.725‐2.542, P = 0.034) for dIAD ≥20 mm Hg and 1.702 (95% CI1.025‐2.828, P = 0.040) for dIAD between 15 and 19 mm Hg, and the multivariate adjusted OR of coronary heart disease prevalence was 1.726 (95% CI 1.093‐2.726, P = 0.019) for dIAD ≥20 mm Hg and 1.498 (95% CI 0.993‐2.261, P = 0.044) for dIAD between 15 and 19 mm Hg. The relationship between cardio‐cerebral vascular disease and dIAD was significant in a Chinese community population. Further cohort studies are needed to investigate the association of different levels of IAD with the incidence of cardiovascular and cerebrovascular diseases and subsequent mortality.     

急性脑梗死患者血清AQP1、AQP4、VEGF表达与脑水肿严重程度的关系

目的探究急性脑梗死患者血清水通道蛋白1(AQP1)、水通道蛋白4(AQP4)、血管内皮生长因子(VEGF)表达与脑水肿严重程度的关系。方法选取急性脑梗死患者78例为观察组,另选取健康者80例为对照组,采用酶联免疫吸附试验检测血清AQP1、AQP4、VEGF水平,应用无创脑水肿动态监护仪测定电阻抗扰动系数值判断脑水肿严重程度,根据电阻抗扰动系数将观察组分为重度组(9.5)及轻度组(7.5电阻抗扰动系数≤9.5),分析其血清AQP1、AQP4、VEGF表达与脑水肿严重程度的相关性,应用Logistic回归模型对影响脑水肿严重程度的因素进行分析。结果观察组血清AQP1、AQP4、VEGF水平与电阻抗扰动系数均显著高于对照组(P0.05);血清AQP1、AQP4、VEGF水平均与电阻抗扰动系数呈显著正相关(均P0.05);重度组血清AQP1、AQP4、VEGF均显著高于轻度组(P0.05);高血压、AQP1、AQP4、VEGF是影响脑水肿严重程度的危险因素(均P0.05)。结论急性脑梗死患者血清AQP1、AQP4、VEGF表达异常,呈显著升高趋势,并与脑水肿形成密切相关,其表达水平越高脑水肿程度越严重。     

Risk of variant Creuzfeldt–Jakob disease from factor concentrates: current perspectives

The demonstration of iatrogenic transmission of Creuzfeldt-Jakob disease (CJD) through therapeutic interventions led to substantial concerns in communities requiring blood products in the 1980s and 1990s. These concerns led some regulatory authorities to adopt a very precautionary approach and require recall of plasma products, including factor concentrates, which included donors at risk of CJD. The FDA's approach on recall contributed to a substantial lack of plasma products on the world market in the mid- to late 1990s. Growing epidemiological evidence of non-transmission of CJD to humans through blood, as well as demonstration of the plasma fractionation system's ability to eliminate CJD-type agents, led the FDA to rescind its measures for product recall in 1998. Although no evidence exists that the variant strain of CJD (vCJD) will behave any differently to the classic strain (cCJD) of the disease during fractionation, indications that higher levels of the strain may be present in the blood in vCJD, as well as the uncertainty regarding the epidemiology of the disease, has led to a new round of precautionary measures aimed at minimizing vCJD risk. Currently, the traditional approach to product safety through appropriate donor selection, screening using laboratory tests and systematic pathogen elimination is not completely possible for addressing the risk of vCJD. The only definite risk factor for vCJD is residence in a country where meat products from cattle with bovine spongiform encephalopathy (BSE) have been consumed; currently this is predominantly the United Kingdom but the appearance of BSE in other European countries has stimulated non-European regulatory authorities to defer blood donors from most of Europe. There is currently no screening test available for vCJD. Plasma fractionation techniques fortuitously appear to eliminate substantial amounts of vCJD like agents but only one pathogen eliminating technique, nanofiltration, has been proposed for specifically eliminating vCJD-like agents. Despite the current uncertainty, it is possible to be cautiously optimistic regarding the safety of factor concentrates from the risk of vCJD. An accumulating body of evidence suggests that it is unlikely that the plasma pool from countries with moderate BSE epidemics will contain sufficient levels of vCJD agent to lead to an infective final product. Nevertheless, the development of a blood screening test and more dedicated elimination methods are high priorities for the blood industry as it faces this new threat. The community of blood product users, including people with haemophilia, need to be in a position to make an informed choice regarding the risk of this new agent. Such a choice needs to take into account the alternatives to plasma product therapy such as recombinant concentrates and the risks to product supply ensuing from an excessive reliance on one form of product.