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To cite this article: Chandler WL, Yeung W, Tait JF. A new microparticle size calibration standard for use in measuring smaller microparticles using a new flow cytometer. J Thromb Haemost 2011; 9: 1216-24.Summary. Background: Microparticle size measurements are often calibrated on flow cytometers using polystyrene microspheres that forward scatter more light vs. particle diameter than cellular microparticles. Methods: We compared theoretical with measured forward angle light scattering on the LSRII, FC500 and Apogee A40 using polystyrene and silica microspheres vs. synthetic lipid vesicles and platelets, then compared plasma microparticle counts using different calibration strategies. Results: Polystyrene and silica microspheres with higher refractive indices forward scattered more light with a wavelength of 488 nm for a given size microparticle than did lipid vesicles or platelets. The LSRII and FC500 did not count many, and were unabletoseparatebysize,polystyrenemicrospheres< 0.5 lmin diameter. On the Apogee A40, polystyrene microspheres could be separated by size down to 0.2 lm, and a polystyrene microsphere 0.4 lm in diameter produced the same forward scatter relative intensity as a 1-lm lipid or cellular microparticle. Using the new calibrator, the Apogee A40 found 80 000-4 000 000 lL )1 total microparticles, 11 000-350 000 lL )1 annexin V positive microparticles and 6000-350 000 lL )1 platelet microparticles < 1 lm in plasma samples. Conclusions: The Apogee A40 was able to resolve size differences in polystyrene microspheres down to 0.2 lm and microparticles down to 0.4 lm. On the Apogee A40 we propose using a 0.4-lm polystyrene microsphere as equivalent to a 1-lm cellular microparticle for size calibration. Using this calibrator, the Apogee A40 detected higher numbers of total, platelet and annexin V positive microparticles than were found using a Megamix gate.

Previous studies have shown that overall fibrinolytic activity in blood follows a diurnal rhythm with a peak in the morning and a trough in the evening. The purpose of this study was to determine which fibrinolytic factor(s) was responsible for this diurnal rhythm. Resting and postvenous occlusion tissue-type plasminogen activator (t-PA) activity, resting t-PA antigen, and resting plasminogen activator inhibitor 1 (PAI-1) activity were measured in the morning and evening in 33 healthy men (mean age, 31 years) and in 15 patients (mean age, 57 years) with previous myocardial infarction or unstable angina. PAI-1 activity and t-PA antigen were significantly higher (p <0.01) in the morning compared with the evening in controls and patients. In contrast, resting t-PA activity was significantly lower in the morning (p <0.01) in both groups and was inversely correlated with PAI-1 activity (r= -0.57, p < 0.0001). Postvenous occlusion t-PA activity and t-PA capacity were not significantly different between morning and evening in either group. Because t-PA antigen levels and PAI-1 activity were highest in the morning, the variation in t-PA activity was probably not due to decreased secretion of t-PA but instead to changes in the secretion of PAI-1. Our findings indicate that diurnal variations in PAI-1 activity may reduce fibrinolytic activity in the morning in healthy individuals and in patients with coronary artery disease. (Circulation 1989;79:101-106) I ncidence of acute myocardial infarction follows a circadian rhythm with a peak in the morning and a trough in the evening. Muller et all found a threefold increase in the frequency of myocardial infarction at 9 AM compared with 11 PM. A similar diurnal variation in the time of onset has been found for a number of other disorders associated with arterial thrombosis, including sudden cardiac death, angina at rest, and stroke.2-4A number of different factors may play a role in the circadian variation of arterial thrombosis. Heart rate, arterial blood pressure, and catecholamine levels have been shown to peak in the morning hours, indicating that coronary vasoconstriction may be increased in the morning as well.5-7Another approach has been to study factors that may increase the risk of initiating thrombus formation. Platelet aggregability has been found to increase in the morning after arising.8,9 A concurrent increase in From plasma epinephrine and norepinephrine were also found. These studies have concentrated on factors that may increase the risk of vasoconstriction or thrombus initiation. Another possible explanation would be alterations in blood components that depress the rate of thrombus removal. In vivo, newly forming thrombi are removed by the fibrinolytic system. Fibrinolysis is initiated by tissue-type plasminogen activator (t-PA), an enzyme secreted by endothelial cells that, in the presence of fibrin, converts the proenzyme plasminogen into its active form, plasmin.10 In turn, plasmin proteolytically degrades the fibrin that holds the thrombus together. Th...

Thrombin generation studies indicate that Trauma with ACT patients show dysregulated hemostasis characterized by excessive non-wound-related thrombin generation due to a combination of circulating procoagulants capable of activating coagulation systemically and reduced inhibitor levels allowing systemic thrombin generation to continue once started.

Women going through the menopausal transition have deleterious changes in inflammatory markers and adipokines that correlate with increased visceral adiposity.

Cardiopulmonary bypass (CPB) is a unique clinical scenario that results in widespread activation of the hemostatic system. However, surgery also results in normal increases in coagulation activation, platelet activation, and fibrinolysis that are associated with normal wound hemostasis. Conventional CPB interferes with normal hemostasis by diluting hemostatic cells and proteins, through reinfusion of shed blood, and through activation on the bypass circuit surface of multiple systems including platelets, the kallikrein-kinin system, and fibrinolysis. CPB activation of the kallikrein-kinin system increases activated factor XIIa, kallikrein, bradykinin, and tissue plasminogen activator levels, but has little effect on thrombin generation. Increased tissue plasminogen activator and circulating fibrin result in increased plasmin generation, which removes hemostatic fibrin. The nonendothelial surface of the bypass circuit, along with circulating thrombin and plasmin, lead to platelet activation, platelet receptor loss, and reduced platelet response to wounds. In this review, we highlight the major mechanisms responsible for CPB-induced activation of the hemostatic system and examine some of the markers described in the literature. Additionally, strategies used to reduce this activation are discussed, including limiting cardiotomy suction, increasing circuit biocompatibility, antithrombin supplementation, and antifibrinolytic use. Determining which patients will most benefit from specific therapies will ultimately require investigation into genetic phenotypes of coagulation protein expression. Until that time, however, a combination of approaches to reduce the hemostatic activation from CPB seems warranted.

Hemolytic uremic syndrome (HUS) usually occurs after infection with Shiga toxin-producing bacteria. Thrombotic thrombocytopenic purpura, a disorder with similar clinical manifestations, is associated with deficient activity of a circulating metalloprotease that cleaves von Willebrand factor at the Tyr842-Met843 peptide bond in a shear stress-dependent manner. We analyzed von Willebrand factor-cleaving metalloprotease activity and the status of von Willebrand factor in 16 children who developed HUS after Escherichia coli O157:H7 infection and in 29 infected children who did not develop this complication. Von Willebrand factor-cleaving metalloprotease activity was normal in all subjects, but von Willebrand factor size was decreased in the plasma of each of 16 patients with HUS. The decrease in circulating von Willebrand factor size correlated with the severity of thrombocytopenia and was proportional to an increase in von Willebrand factor proteolytic fragments in plasma. Immunohistochemical studies of the kidneys in four additional patients who died of HUS demonstrated glomerular thrombi in three patients, and arterial and arteriolar thrombi in one patient. The glomerular thrombi contained fibrin but little or no von Willebrand factor. A decrease in large von Willebrand factor multimers, presumably caused by enhanced proteolysis from abnormal shear stress in the microcirculation, is common in HUS. HUS, characterized by acute renal failure, hemolysis with schistocytes on blood smears, and thrombocytopenia, is accompanied by thrombotic microangiopathy of the kidneys and of other organs (1). The syndrome covers a diverse spectrum of microangiopathic disorders (2-4), but most cases occur after infection with Shiga toxin-producing bacteria, such as Escherichia coli O157:H7 (5) or Shigella dysenteriae serotype 1 (6).TTP, a disorder with some clinical, laboratory, and histopathologic similarities to HUS, has been associated with abnormal homeostasis of von Willebrand factor, a protein that is secreted from endothelial cells as a disulfide-linked polymer of a polypeptide with 2050 amino acid residues. Circulating von Willebrand factor is normally cleaved between Tyr842 and Met843 (7) in a shear stress-dependent manner (8) by a plasma metalloprotease (9, 10), generating a series of multimers. Without this metalloprotease activity, von Willebrand factor, when unfolded by shear stress (11), has increased platelet-aggregating activity (12). It is postulated that this increased activity facilitates the formation of arteriolar and capillary platelet thrombi in TTP. Indeed, acquired TTP has been associated with deficient von Willebrand factor-cleaving metalloprotease activity caused by inhibitory antibodies (12, 13).

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